SV40 expression vector containing HBxAg as an expression marker

ABSTRACT

Cloning and expression vectors for hepatitis B HBxAg, cell cultures containing those vectors, and diagnostic systems and methods for assaying for the presence of HBxAg and anti-HBxAg in a body sample are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of copending application Ser. No. 587,570filed on Mar. 8, 1984, abandoned.

DESCRIPTION

1. Technical Field

The present invention relates to recombinant DNA technology, expressionvectors, and polypeptide expression markers, and more specifically tothe cloning of the gene for hepatitis B HBxAg, an expression vectorcontaining HBxAg as well as assay systems and methods for determiningthe presence of HBxAg and anti-HBxAg in body samples.

2. Background Art

A. Cloning and Vectors

The introduction of exogenous DNA into eucaryotic cells has become oneof the most powerful tools of the molecular biologist. This processrequires efficient delivery of the DNA into the nucleus of the recipientcell and subsequent identification of cells that are expressing theforeign DNA.

Engineered vectors such as plasmids or bacteriophages (phages) or otherDNA sequeuce that is able to replicate in a host cell can be used toconstruct cells that act as factories to produce large amounts ofspecific viral proteins. Recombinant plasmids will be used herein asexemplary vectors, also called cloning vehicles. See U.S. Pat. No.4,338,397, incorporated herein by reference.

Plasmids are extrachromosomal genetic elements found in a variety ofbacterial species. They are typically double-stranded, closed, circularDNA molecules. The most widely used plasmid is pBR 322, a vector whosenucleotide sequence and endonuclease cleavage sites are well known.

Nucleic acid production using plasmid or phage vectors has become verystraightforward. The plasmid or phage DNA is cleaved with a restrictionendonuclease and joined in vitro to a foreign DNA of choice. Theresulting recombinant plasmid or phage is then introduced into a cellsuch as E. coli, and the cell so produced is induced to produce manycopies of the engineered vector. Once a sufficient quantity of DNA isproduced by the cloning vector, the produced foreign DNA is excised andplaced into a second vector to produce or transcribe the protein orpolypeptide encoded by the foreign gene.

Depending on the DNA (intact gene, cDNA, or bacterial gene), it may benecessary to provide eucaryotic transcription and translation signals todirect expression in recipient cells. These signals may be provided bycombining the foreign DNA in vitro with an expression vector.

Expression vectors contain sequences of DNA that are required for thetranscription of cloned genes and the translation of their messengerRNA's (mRNA's) into proteins. Typically, such required sequences orcontrol elements are: (1) a promoter that signals the starting point fortranscription; (2) a terminator that signals the ending point oftranscription; (3) an operator that regulates the promotor; (4) aribosome binding site for the initial binding of the cells' proteinsynthesis machinery; and (5) start and stop condons that signal thebeginning and ending of protein synthesis.

To be useful, an expression vector should possess several additionalproperties. It should be relatively small and contain a strong promoter.The expression vector should carry one or more selectable markers toallow identification of transformants. It should also contain arecognition site for one or more restriction enzymes in regions of thevector that are not essential for expression.

The construction of expression vectors is, therefore, a complicated andsomewhat unpredictable venture. The only true test of the effectivenessof an expression vector is to measure the frequency with which thesynthesis of the appropriate mRNA is initiated. However, quantitation ofmRNA is tedious, and it is often difficult to obtain accuratemeasurements. Other more practicable means have, therefore, beendeveloped to detect transformation.

One such means has been to monitor synthesis of foreign proteins intransformed cells with enzymatic assays. Several marker genes have beendeveloped for indicating that transformaion has occurred.

Another means used to monitor transformation involves the use ofimmunological reagents. If the level of expressed protein issufficiently high, then cytoplasmic or surface immunofluorescence withan antibody conjugated to a fluorescent dye such as fluorescein orrhodamine may be used to detect vector-specific protein expressionproducts.

More commonly, transformed cells are cultured in the presence ofradioactivity after immunoprecipitation. This approach has usedStaphyloccocus aureus protein A selection of immune complexes [Kessler,(1975), J. Immunol., 115:1617-1624] and the Western blotting procedure[Renart et al., (1979), Proc. Natl. Acad. Sci. USA, 76:3116-3120] todetect transformation-specific markers.

Analysis of gene expression using Simian Virus 40 (SV40) vectors is byfar the most explored eucaryotic transformation technique at thebiological and immunochemical levels. Genetic and biochemicalinformation relating to the organization of the SV40 genome has beenestablished or confirmed by the nucleotide sequence of the viral genome.Review by Tooze (1980), Molecular Biology of Tumor Viruses, 2nd ed.,Part 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Thedesign of different SV40 vector molecules has relied on the accuratemapping of genetic signals and the use of restriction endnucleases forthe isolation of defined fragments from the SV40 genome.

SV40 was developed initially as a eucaryotic-transducing vector using alytic system. Mulligan et al., (1979), Nature (London) 277:108-114.Subsequently, transforming (nonlytic) vectors were constructed withisolated segments of the SV40 genome. Review by Elder et al., (1981),Annu. Rev. Genet., 15:295-340.

Hamer et al. were the first to suggest that SV40 might be used to clonegenes for which no probe was available. They suggested double-strandedcDNA copies from a heterogeneous mRNA population could be "shotgunned"into an SV40 vector, and virus carrying the desired sequence could beidentified by using a radioactive or fluorescent antibody.

Hamer et al. first reported the construction of an SV40 recombinantexpression vector containing an expression marker in 1979. Hamer et al.,(1979), Cell, 17:725-735. Their SV40 vector contained the viral DNAsequences from the BamHI endonuclease restriction site at 0.14 map unitsclockwise to the HaeII restriction site at 0.82 map units. In additionto the entire early gene A and the origin of viral DNA replication, thevector contained the viral promoter, leader, intervening sequence, 5'portion of the body and 3' terminal sequences for the viral late 195mRNA. It did not contain 1660 base pair (bp) of late region sequencesencoding the viral protein UPI, 2 and 3. Priers et al., (1978), Nature,273:113-120 and Reddy et al., (1978), Science, 200:494-502.

Rabbit beta-globlin gene coding sequences were ligated into the abovevector as an expression marker. To determine whether rabbit beta-globinwas being synthesized in monkey cells infected with their recombinantvector, Hamer et al., supra, used a radioimmunoassay capable ofdetecting as little as 1.0 nanogram of globin.

Although Hamer et al. were able to demonstrate positive evidence ofbeta-globin expression, they expressed several reservations as to theutility of the SV40/beta-globin recombinant system. First, since globinis only sparingly soluble, significant losses may have been sustainedduring the preparation of samples for measurement. Thus, thedetermination of the amount of globin in the infected cells may be inerror by as much as 10-fold. Second, the assay cannot distinguishbetween authentic globin and other immunologically- related products,such as read-through protein or polypeptide fragments.

A factor that Hamer et al. did not address is the high degree ofhomology between all eucaryotic globins. This homology makes itdifficult to distinguish vector-induced globin expression from globinendogenous to the host cell system.

B. Hepatitis B Virus Peptides and Anti-Polypeptide Antibodies

The hepatitis viruses are markedly different agents. They are groupedtogether strictly by virtue of the "target" organ the affect, the liver.Although a number of viruses affect the liver as part of systemicinfections, the term "hepatitis viruses" is usually taken to mean Type A(HAV), Type B (HBV), and the non-A, non-B agents. Of the three types ofviruses, HBV is by far the most explored at the biological,immunochemical, and clinical levels.

HBV is classified as a DNA virus and differs in many respects from allother families of DNA viruses. HBV is composed of an outer coat (moresubstantial than a membrane or envelope) consisting of protein, lipid,and carbohydrate, and bearing a unique antigen complex; i.e., thehepatitis B surface antigen (HBsAg). It also contains aninner-nucleocapsid with an antigenic specificity distinct from that ofthe surface antigen; i.e., the hepatitis B core antigen (HBcAg).

A soluble antigen, HBeAg, is also recognized in the art. This antigen isthought to consist of HBcAg polypeptides that are not assembled into HBVcores, and consequently have a unique antigenic specificity in theunassembled state.

In typical self-limiting acute HBV infections, the following serologicalmarkers appear sequentially in serum of an infected host: HBsAg, HBeAg,anti-HBC, anti-HBE, and anti-HBS. The appearance of anti-HBE signals theeventual loss of detectable HBsAg. This is true in all cases ofself-limited acute HBV infection. Following the disappearance of HBS,there is a delay of from a few weeks to several months before theappearance of anti-HBS.

During chronic HBV infection, HBsAg and anti-HBC are present. The host'sserum can show either the HBeAg or anti-HBE serological markers; i.e.the patient can either be HBeAg or anti-HBE positive.

Sensitive and specific radioimmunoassays and enzyme immune assays forseveral of the HBV markers are in wide use. These highly sensitiveserologic tests have provided a basis for monitoring the appearance ofvirus and immune response markers during the course of HBV infection.

In the past few years, many studies have indicated that each serologicmarker signifies specific viral events for host responses during HBVreplication. The profile of serologic markers at various stages duringthe clinical course of disease can thus offer useful diagnostic andprognostic information.

The association between hepatitis B virus and humanhepatocellular-carcinoma (HCC), liver cancer, has been extensivelystudied, and seroepedemiological as well as histopathological findingsstrongly suggest that HBV is directly or indirectly involved in theetology of liver cancer. A number of hepatoma cell lines have beenderived from human HCC, and detection of HBV-specific DNA integratedinto the genome of two such cell lines, PLC/PRF/5 and EPH3B, have beenreported. However, in these cell lines, only the hepatitis B surfaceantigen has been expressed in tissue culture as a virus-specific geneproduct. Other markers of HBV such as hepatitis B core antigen,hepatitis BE antigen, and a DNA polymerase have not been detected.

Some DNA tumor viruses of animals can produce transformation through theaction of viral genes that regulate the replication and integration ofthe viral genome, and transformed cells by such viruses can bear a T(tumor) or neo(new) antigen expressed by the transforming genes. Recentevidence for an antigen analogous to T antigen has been obtained inhuman hepatoma cells containing integrated HBV genes using theanti-complement immunofluorescent staining technique. This antigen hasbeen designated HBV-associated nuclear antigen (HBNA). Wen et al.,(1983) Infect. Immun., 39:1361-1367.

HBNA was detected in sera from several HBsAg-positive HCC patients, andexpression of the antigen was demonstrated in both cell culture andtumor tissue. In addition, anti-HBNA antibodies were found in the seraof some HBsAg-positive patients with HCC. HBNA may represent thepreviously unrecognized expression of an HBV gene.

In 1979 Galibert et al., Nature, 281:646-650, reported the nucelotidesequence of the HBV genome, a circular DNA of about 3200 bases that ispart double- and part single-stranded, a feature unique among viruses.The long or L strand (completely circular) of the genome was found tocontain four reading frames large enough to account for viral proteins.These regions were termed S, C, P and X, and are shown schematically inFIG. 1.

Regions S and C have been found to contain the genes for HBsAg and HBcAgrespectively. Region P is thought to code for a protein similar in sizeand amino acid residue content to a DNA polymerase. Region X waspostulated by Wen et al., supra, to be one of the probable sources ofthe gene coding for HBNA.

The mere tentative assignment of functions for the genes in regions Pand X demonstrates the gap that still exists in understanding thegenetic organization and molecular biology of HBV. A reason for this gaphas been the absence of an in vitro system for propagation of the virus.

Until recently, the only source of HBV was the serum of human patients.The failure of attempts to grow the virus in cell culture is the resultof its very narrow host cell range.

One approach to the problem of producing HBV DNA and its gene productshas been to use recombinant DNA technology. This technology enables thelarge scale production of the nucleic acid sequences that code for aparticular viral protein.

Tiollais et al. (1981), Science, 213:406-411 reported transformation ofE. coli with pBR322 containing the gene coding for HBsAg, and reportedproduction of significant quantities of that isolated gene. Thoseworkers also wanted to study the HBsAg gene's location within the HBVgenome, and the factors that affected its expression into protein. To dothis they constructed expression vectors containing the HBsAg gene foruse in both bacterial and mammalian cells.

One of the expression vectors constructed by Tiollais et al., supra,achieved bio-synthesis of a protein in E. coli that contained HBsAgantigenic determinants. It was built by inserting a portion of the genecoding for HBsAg into the bacteriophage plac5-1UV5 so as to conserve thereading frame of natural HBV.

In order to study HBV gene expression in mammalian cells Tiollais etal., supra, constructed a series of HBsAg-expression plasmids byinserting transfection elements at various locations within the wholeHBV genome. The transfection elements allowed the entire HBV genome tobe integrated in several different orientations into the genome of mousecells transformed with the vector. In creating the vectors in this waythose workers attempted to use HBV's naturally occuring genetic controlelements.

Only three of the six expression vectors reported by Tiollais et al.,supra, produced expression of the desired HBsAg protein. Its productionwas detected by testing for its presence in tissue culture fluids usingsheep anti-HBsAG antiserum. The other viral markers known at the time ofthe study (i.e., HBcAg, HBeAg and DNA polymerase) were not detected inthe transformed cells.

At the time of the above Tiollais et al. study, the possible existenceof Region X was known as was the possibility that it may code for a HBVprotein. That is, it was known that the HBV genome contained a capacityto code for more proteins than had been previously associated with thevirus.

This problem has been called genotype in search of phenotype. It is thisproblem, inter alia, that Wen et al., supra, were addressing when theypostulated Region X as containing the gene coding for HBNA.

Prior to the above Tiollais et al. and Wen et al. studies, Sutcliffe etal., (1980) Nature, 287:801-805, demonstrated, inter alia, a generaltechnique for solving this problem. They chemically synthesized apolypeptide from within the protein predicted by the nucleotide sequenceof a viral gene whose protein product was unknown. Antibodies wereraised to the polypeptide and were reacted against all the proteins madeby cells infected with the virus. Using antibodies to portions of apredicted protein, Sutcliffe et al. detected a previously unknown andunrecognized viral protein product.

To date, the use of this or any other technique to unequivocallyidentify the protein product of HBV genome Region X has not beenreported. This may be because while the general concept of preparingsynthetic antigens and using them to induce antibodies of predeterminedspecificity has been described, there remains a large area of thistechnology that continues to defy predictability.

The reasons for this are several. First, protein amino acid residuesequences deduced from a genetic sequence are of a hypothetical natureunless the nucleotide sequence reading frame is firmly establishedbecause of the redundancy of the genetic code.

Second, a synthetic antigen does not necessarily induce antibodies thatimmunoreact with the intact protein in its native environment. Third, ahost's natural antibodies to an immunogen rarely immunoreact with apolypeptide that corresponds to a short linear portion of theimmunogen's amino acid sequence.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates several aspects. One aspect is arecombinant DNA comprising an exprssion vector linked to the gene codingfor HBxAg. In a particularly preferred recombinant DNA, the DNAexpression vector comprises a first DNA sequence comprising the SimianVirus 40 viral DNA sequence from the BamHI endonuclease restriction siteat base position 2468 clockwise to the HaeII endonuclease restrictionsite at base position 767 according to FIG. 2, and the gene coding forHBxAg is provided by a second gene DNA sequence comprising the hepatitisB virus DNA sequence from the HaeII endonuclease restriction site atbase position 1437 clockwise to the BamHI endonuclease restriction siteat base position 28 according to FIG. 1. The first and second DNAsequences are operatively linked to promote expression of HBxAg.

Another aspect of this invention is constituted by a recombinant DNAthat comprises a gene coding for HBxAg or for a polypeptide constitutinga substantial portion of HBxAg. A particularly preferred recombinant DNAcontains the gene coding for HBxAg and has the base sequence shown inFIG. 1 as covering base positions 1437 clockwise to 28, or a substantialportion thereof.

A plasmid coding for at least one recombinant DNA that comprises a genecoding for HBxAg or for a polypeptide constituting a substantial portionthereof constitutes another aspect of this invention. That plasmid mayinclude the base sequence shown in FIG. 1 as covering base positions1437 clockwise to 28, or a substantial portion thereof. In particularlypreferred practice, the plasmid comprises a first DNA sequencecomprising the Simian Virus 40 (SV40) viral DNA sequence from the BamHIendonuclease restriction site at base position 2468 clockwise to theEcoRI endonuclease restriction site at base position 1717 according toFIG. 2; a second DNA sequence comprising the plasmid pBR322 DNA sequencefrom the BamHI endonuclease position at base position 375 clockwise tothe EcoRI endonuclease restriction site at base position zero accordingto FIG. 3; and a third DNA sequence comprising the hepatitis B virus DNAsequence from the BamHI endonuclease restriction site at base position1400 clockwise to the BamHI endonuclease restriction site at baseposition 28 according to FIG. 1. In that plasmid, the first and secondDNA sequences are operatively ligated at their respective EcoRIendonuclease restriction sites; the second and third DNA sequences areoperatively ligated at their respective BamHI endonuclease restrictionsites; and the first and third DNA sequences are operatively ligated attheir respective BamHI endonuclease restriction sites according to FIG.4.

A method of producing HBxAg or a substantial polypeptide portion ofHBxAg constitutes another aspect of this invention. According to thisaspect, a host containing an expression system of this invention isgrown in a culture broth medium until HBxAg or a substantial polypeptideportion thereof is produced and accumulated in the host or culturebroth. The accumulated HBxAg or the substantial polypeptide portionthereof is thereafter recovered.

Yet another aspect of this invention contemplates an antigenic syntheticpolypeptide. That synthetic polypeptide contains about 6 to about 40amino acid residues and corresponds in sequence to the sequence of anantigenic determinant of HBxAg. A particularly preferred polypeptideincludes an amino acid residue sequence selected from the group ofpolypeptide sequences represented by the formulae written from left toright and in the direction of amino-terminus to carboxy-terminus

(i) Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp;

(ii) Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val;and

(iii) Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala.

A water-soluble or water-dispersible antigenic polymer containing aplurality of joined synthetic polypeptide repeating units bondedtogether by oxidized cysteine residues is also contemplated herein. Therepeating units are comprised of the polypeptides discussed hereinabovethat contain cysteine residues at both the amino- and carboxy-terminii,or contain one cysteine residue at one terminus and one cysteine residuewithin the polypeptide chain. Particularly preferred polypeptiderepeating units, including the amino- and carboxy-terminal cysteineresidues, are represented by a formula, written from left to right andin the direction from amino-terminus to carboxy-terminus, selected fromthe group consisting of

Cys-Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp-Cys;

Cys-Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val-Cys;and

R¹ -Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala-R²,

wherein each of R¹ and R² is a cysteine residue, with the proviso thatonly one of R¹ and R² is present.

Receptor molecules that include an antibody combining site such asantibodies capable of immunoreacting with one of the before-mentionedantigenic polypeptides are also contemplated herein. The particularlypreferred polypeptides with which these receptor molecules immunoreactare illustrated by the polypeptides described hereinbefore. Thesereceptor molecules also immunoreact with HBxAg or with a substantialpolypeptide portion thereof.

A diagnostic assay system for determining the presence of a detectableamount of HBxAg in a body sample to be assayed is also contemplated.This system comprises at least one container that contains a reagentthat includes the above-described receptor molecules. This system mayfurther include a second reagent in a second container, which secondreagent is the antigenic synthetic polypeptide with which the receptormolecules immunoreact. A means for signalling the immunoreaction betweenthe receptor molecules and HBxAg may constitute a further part of thediagnostic assay system.

A method for assaying for the presence of a detectable amount of HBxAgin a body sample constitutes a further aspect of this invention. Here,proteins from a body sample to be assayed for the presence of adetectable amount of HBxAg are admixed with the above receptor moleculesin the presence of an indicating means for signalling an immunoreactionbetween the receptors and HBxAg. The admixture is maintained for a timeperiod sufficent for the indicating means to signal that animmunoreaction has occurred. The presence of that signal is thenascertained.

BRIEF DESCRIPTION OF THE DRAWINGS

In the Figures forming a portion of the disclosure of this invention:

FIG. 1 is a schematic diagram showing the physical structure and geneticorganization of the HBV/adw genome from Tiollais et al. (1981), Science,213:406-411 as modified by Ono et al. (1983), N.A.R., 11:1747-1757. The5'-end of the long (L) strand is reported to be base paired with the5'-end of the short (S) strand. Certain restriction sites indicated byarrows linked to arcs and the abbreviation for the restrictionendonuclease correspond to the physical map of the HBV/adw genomeanalyzed by Ono et al., supra. The broad arrows surrounding the genomecorrespond to the four large, open regions of the L strand. These fourpotential coding regions are designated S, P, X and C. The number ofamino acid residues in parentheses (aa) following each of the fourdesignated regions corresponds to the length of the hypotheticalpolypeptide encoded by each region. The two regions corresponding to thedefined genes S and C are indicated by dotted areas within the broadarrows. The single EcoRI endonuclease cleavage site is used as the pointof origin of the map.

FIG. 2 schematically illustrates an SV40 DNA restriction map preparedfrom the primary sequence published by Reddy et al. (1978), Science,200:494-501, as modified by Van Heuverswyn and Fiers (1979), Eur. J.Biochem., 100:51-60.

FIG. 3 schematically illustrates a plasmid pBR322 restriction mapprepared from primary sequence data of Sutcliffe (1979), Cold SpringHarbor Symposium on Quantitative Biology 43, 77.

FIG. 4 schematically illustrates the method, described in the Materialsand Methods section hereinafter, used for inserting DNA fragments intovectors of this invention made from AM6 (wavy lines), pBR322 (solidlines) and SV40 (dashed lines) to produce the cloning vector SVAM191 andthen the expression vector SVHBV-3 that expresses a substantial portionof HBxAg. Relative positions of restriction endonuclease sites forBamHI, EcoRI and HaeII are illustrated by closed circles, open circlesand closed triangles, respectively. The origin (ori) and early and latepromoters of the SV40 genome are also indicated.

FIG. 5 depicts a linear portion of the SVHBV-3 recombinant expressionvector containing the genes for the SV40 late promotor, theamino-terminal 99 amino acid residues (99aa), and a substantialpolypeptide portion (132aa; 132 of 154 amino acid residues) of the HBxAggene sequence coding for the 132 carboxy-terminal amino acid residues.Also depicted is the mRNA for the VP2 HBxAg fusion protein expressed bythe vector.

FIG. 6 illustrates the translated amino acid residue sequence shown fromleft to right and in the direction from amino-terminus tocarboxy-terminus of the gene coding for HBxAg. The substantial portionof HBxAg expressed by SVBHV-3 is illustrated by the arrow at amino acidresidue position 23 (Ala), which is the amino-terminal residue of theexpressed HBxAg polypeptide. The relative position of syntheticpolypeptides designated 99, 100 and 142 of this invention in the HBxAgsequence are illustrated by the three labeled, underlined amino acidresidue sequences. The amino acid residue sequence of syntheticpolypeptide 99 therefore corresponds to positions 100 through 115 fromthe amino-terminal Met residue shown in the Figure, the sequence ofsynthetic polypeptide 100 corresponds to positions 115 through 131 fromthe amino-terminal Met residue of the Figure, and the sequence ofsynthetic polypeptide 142 corresponds in sequence to positions 144through 154 from the amino-terminal Met residue of the Figure; i.e., theeleven carboxy-terminal residues.

FIG. 7 is a photograph of an autoradiogram showing the reactivity ofanti-X antisera with two human hepatoma cell lysates. Two human hepatomacell lines, PLC/PRF/5 and HepG2 were grown in Dulbecco's Modification ofEagle's Medium (DMEM) with 10% fetal calf serum to subconfluency (day 6of a 1:5 split from a confluent culture). Supernatants were removed, theflasks containing the monolayers were placed on ice for 5 minutes beforethe cells were collected by scraping, and the collected cells werepelleted by centrifugation. Cell pellets were washed with phosphatebuffered saline (PBS), quick frozen at -70° C. and lyophilizedovernight. The resultant cell powders were dissolved in PBS and broughtto a final concentration of 2 milligrams per milliliter (mg/ml) insample buffer. The samples were boiled for 5 minutes, and cell debriswas removed by centrifugation in a Beckman microfuge for 30 minutes.Fifty micrograms of cell lysate were incubated for 15 minutes in RIPAbuffer [PBS, containing 1% Nonidet P-40 (polyoxyethylene (9) octylphenyl ether), 0.05% sodium deoxycholate and 0.1% SDS] and radiolabeledwith 3 microCuries of ¹²⁵ I by the chloramine T reaction McConahey etal. (1966), Int. Arch. Allergy 9 185-189. 1.5×106 Counts per minute(cpm) of each of the radiolabeled hepatoma lysates were reacted with 10microliters of anti-X polypeptide for 60 minutes in RIPA. Theantigen-antibody complexes (immunoreaction-products) were precipitatedwith formalin-fixed Staphylococcus aureus. The pellets were washed oncewith RIPA and twice with 500 millimolar (mM) LiCl, 100 mM Tris (pH=8.5),and were analyzed for radioactivity. All pellets were dissolved in 40microliters sample buffer [0.0625M Tris-HCl (pH 6.8), 2% SDS, 10%glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue], boiled for3 minutes, centrifuged to remove cell debris and subjected to SDS-PAGEas follows.

The radiolabeled cell lysates were loaded onto denaturing sodium dodecylsulfate-polyacrylamide gels (12.5%) (SDS-PAGE), and subjected toelectrophoresis following the procedures of Laemmli, (1970), Nature,297:680:685. The proteins were electrophoretically transferred tonitrocellulose sheets as described by Towbin et al. (1979), Proc. Natl.Acad. Sci. U.S.A., 76:4350-4354. The nitrocellulose blots were stainedin amido black prior to incubation at 4° C. overnight in BLOTTO [Johnsonet al. (1983), J. Exp. Med., 159:1751-1756] for reduction ofnon-specific binding. Nitrocellulose strips were incubated for 3 hoursat room temperature with a 1:350 dilution of anti-polypeptide antibodiesin a final volume of 10 ml BLOTTO. The strips were washed in BLOTTOprior to a 1 hour incubation with ¹²⁵ I-labeled S. aureus protein A (10⁶cpm per 10 ml). Where competition by a polypeptide is indicated below,the anti-polypeptide antisera were incubated with 100 microgramspolypeptide for 60 minutes prior to the addition of the radiolabeledantigen. The strips were washed as above, rinsed with water, dried andautoradiographed. Lanes 1 and 5 were reacted with anti-polypeptide 99antibodies (anti-99); lane 2 was reacted with anti-99 preincubated withpolypeptide 99; lane 3 was reacted with anti-99 preincubated with anon-related polypeptide; and lane 4 was reacted with a preimmune serumcontrol. Lysates from HepG2 cells lanes 1-4) did not react withanti-polypeptide 99. The numerals on the left-hand margin indicaterelative protein migration positions. Arrows at the right-hand marginindicate the migration positions of proteins having molecular weights of28,000 and 45,000 daltons.

FIG. 8 is a photograph showing the reactivity of anti-X antiserum withchimpanzee (C; lanes 3 and 4) and human (H; lanes 1 and 2) livertissues. Liver sections from chimpanzees and humans were snap frozen inliquid nitrogen and were ground into a fine powder with a mortar andpestle. Cell powders were solubilized in sample buffer, and weresubjected to gel electrophoresis and blotted as described for FIG. 7.Nitrocellulose strips were developed with a 1:50 dilution of antiserumas described in FIG. 7, with two exceptions: (1) 1% bovine serum albumin(BSA) solution was used in place of BLOTTO in all incubations andwashes, and (2) the antigen-bound antibodies were detected withhorseradish peroxidase conjugated goat anti-rabbit IgG. Theantigen-antibody immunoreaction products were visualized by dipping thestrips into the following developing solution. The developing solutionwas prepared from 50 ml of TBS buffer at 37° C. to which were added 250microliters of absolute ethanol containing 8 mg of 4-chloro-1-naphtholand 330 microliters of 30 percent hydrogen peroxide. Antiserum topolypeptide 99 (anti-99) was used in the two left-hand panels, blockedand not blocked, while antiserum to polypeptide 142 (anti-142) was usedin two right-hand panels, blocked and not blocked. Numerals on theleft-hand side of the Figure illustrate gel migration positions forproteins having molecular weights of 66, 45, 31, 21 and 14 kilodaltons,respectively.

FIG. 9 is a photograph of a Southern blot analysis of human andchimpanzee restriction enzyme-cleaved liver DNAs from liver tissues thatexhibited X protein, using hepatitis B virus (HBV) DNA as the bindingprobe. Total DNA was extracted from the cell powders made from thesamples described in FIG. 8. Ten micrograms of DNA were loaded to eachwell of a 1% agarose gel. Restriction enzyme digestion buffers wereaccording to manufacturer (BRL who, where). All digestions were carriedout overnight at 37° C. with 50 units of enzyme (5×DNA concentration inmicrograms). Lanes 1-3 show chimp A-243 liver DNA digested with BamHI,EcoRI or uncleaved, respectively; lanes 4-6 show chimp 344 liver DNAcleaved with BamHI, EcoRI or uncleaved, respectively; lanes 7-10 showhuman HBcAg-positive liver DNA cut with HindIII, BamHI, EcoRI or withoutdigestion, respectively; and lanes 11-14 show human HBsAg-negative liverDNA digested with HindIII, BamHI, EcoRI or undigested, respectively.

FIG. 10 is a pnotograph of an autoradiogram showing the reactivity ofanti-X polypeptide antisera with an X-directed gene product. Confluentmonolayers of BSC-1 cells (2×10⁷ cells) were infected with therecombinant SVHBV-3 stock virus (a mixture of tsA₂₃₉ and SVHBV-3virions) or wild type SV40 [Moriarty et al. (1981), Proc. Natl. Acad.Sci. U.S.A., 78:2606-2610]. The cultures were incubated in serum freeDMEM at 40° C. for 48-72 hours at which time approximately 50%cytopathic effect had occurred. Supernatants were removed, and theflasks containing the monolayers were placed on ice for 5 minutes beforethe cells were collected by scraping. The collected cells werecentrifuged, and the resulting cell pellets were washed with PBS, quickfrozen at -70° C., and lyophilized overnight. The resultant cell powderswere dissolved in PBS and brought to a final concentration of 2 mg/ml insample buffer. The samples were boiled for 5 minutes, and cell debriswas removed by centrifugation in a Beckman microfuge for 30 minutes.Fifty to one hundred micrograms of cell lysate was loaded ontodenaturing polyacrylamide gels (12.5%) and subjected to electrophoresisas beforedescribed. The proteins were electrophoreticallly transferredto nitrocellulose sheets as described previously. The nitrocelluloseblots were stained in amido black prior to incubation at 4° C. overnightin BLOTTO for reduction of non-specific binding. Nitrocellulose stripswere incubated for 3 hours at room temperature with a 1:350 dilution ofanti-peptide antibodies in a final volume of 10 ml BLOTTO. The stripswere washed in BLOTTO prior to a 1 hour incubation with ¹²⁵ I-labelledS. aureus protein A (106 cpm per 10 ml). The strips were washed asabove, rinsed with water, dried and autoradiographed. In cases whereblocking with polypeptide was performed, the antisera were preincubatedwith 100 micrograms of polypeptide solution overnight at 4° C. or 2hours at 37° C. prior to incubation with the nitrocellulose strips.Molecular weights were based on low molecular weights standards(BioRad), and are shown at the left-hand margin of the Figure. Lanes 1and 3 were reacted with anti-polypeptide 99 and anti-polypeptide 142antibodies, respectively. Lanes 2 and 4 were reacted with those sameantibodies preincubated with polypeptide 99 or with polypeptide 142,respectively. Lanes 5 and 6 show the reaction of anti-polypeptide 99 and142 antibodies, respectively, with control cell lysates.

The instant invention has several benefits and advantages. One of thosebenefits is that for the first time, the presence of HBxAg in any cellhas been detected.

Another benefit is that the invention provides an assay method andsystem for detecting the presence of HBxAg in a chronically hepatitisB-infected host animal.

Still another benefit is that use of the invention has enabled thedetection of HBxAg in cells derived from human hepatomas, therebystrengthening the presumed link between hepatitis B virus and this formof cancer.

One of the advantages of the invention is that it provides means forcloning the gene coding for HBxAg.

Another advantage of the invention is that it provides means forexpressing a substantial polypeptide portion of HBxAg that may be usedas an antigen in a diagnostic assay for the presence of antiboies toHBxAg (anti-X antibodies) that are present in the serum of humans.

Yet another advantage of the invention is the preparation of a syntheticpolypeptide whose amino aicd residue sequence corresponds to thesequence of an antigenic determinant of HBxAg that can be used toprepare antibodies that immunoreact with HBxAg as well as withpolypeptides that share an antigenic determinant with HBxAg.

Still further benefits and advantages of the present invention willbecome apparent to those skilled in the art from the detaileddescription and claims that follow.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Transfection: is the acquisition of new genetic markers by incorporationof added DNA in eucaryotic cells.

Transformation: is the acquisition of new genetic markers byincorporation of added DNA in procaryotic cells.

Cloning Vector: is any plasmid or virus into which a foreign DNA may beinserted to be cloned.

Plasmid: is an autonomous self-replicating extrachromosomal circularDNA.

Open Reading Frame: is a DNA sequence which is (potentially)translatable into protein.

Helper Virus: is a virus that provides functions absent from a defectivevirus, enabling the latter to complete the infective cycle during amixed infection.

Gene (Cistron): is the segment of DNA that is involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Expression: the process undergone by a structural gene to produce apolypeptide. It is a combination of transcription and translation.

Clone: describes a large number of identical cells or molecules with asingle ancestral cell or molecule.

Base Pair (bp): is a partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a DNA double helix.

Expression Vector: is any plasmid or virus into which a foreign DNA maybe inserted or expressed.

Downstream: identifies sequences proceeding farther in the direction ofexpression; for example, the polypeptide coding region for a gene isdownstream from the initiation codon.

A. General Discussion

Region X of the hepatitis B virus HBV genome is of interest, inter alia,because recent evidence indicated the possibility of it being expressedand traditional HBV serology had not found its protein product orantibodies to such a product. Recently developed recombinant DNA andsynthetic polypeptide technologies are used herein to overcome some ofthe failures encountered by traditional methodologies with respect tothe expression of HBxAg, and to illustrate the expression of HBxAg incells from human hepatoma-derived cell lines and in liver cells fromchimpanzees and humans having a chronic HBV infection.

To that end, the present invention contemplates cloning and expressionvectors for HBxAg, synthetic polypeptides that correspond to antigenicdeterminants of HBxAg and to antibodies raised to those syntheticpolypeptides that bind to the polypeptides as well as to HBxAg, or to asubstantial portion thereof, as well as assays for the presence ofHBxAg.

The cloning vectors discussed herein are utilized, inter alia, forpreparing useful quantities of an HBxAg-containing gene. That gene inturn may be used, inter alia, to prepare quantities of HBxAg andpolypeptide fragments thereof that may be used in studying hepatitis Bdisease itself.

The novel synthetic polypeptides of this invention are useful, interalia, as antigens in preparing antibodies that immunoreact with HBxAgand substantial polypeptide portions thereof, as well as immunoreactingwith the synthetic polypeptides themselves. The antibodies so preparedmay thereafter be utilized to assay for the presence of HBxAg or asubstantial polypeptide portion thereof in the cells of an infectedanimal host or in tissue culture.

The expression vector may be used to transfect cells and induceexpression of a polypeptide that includes a substantial portion ofHBxAg. The expressed polypeptide may then be utilized as an antigen inan assay for determining the presence of antibodies to HBxAg in a bodysample.

The expression vector and antibodies of this invention may also be usedas a marker for transfected cells that contain the expression vector andfurther include an additional ligated gene whose presence may berelatively difficult to ascertain. Thus, the expression vectordenominate SVHBV-3, described hereinafter, may be opened and ligated tostill another, foreign gene that codes for a protein that may berelatively difficult to assay for. After recircularization, infection ofsuitable cells with the thus formed, new vector, and plating into singlecelled colonies, those cells that incorporated the new vector may beidentified by their expression of the vector-encoded portion of HBxAgfused to the protein encoded by the foreign gene, using the antibodiesof this invention. Such a marker can be particularly useful where thevectoris desired to be expressed in eucaryotic cells; i.e., where drugresistance markers present in bacterial cell vectors such as pBR322 arenot present.

1. Cloning Vectors

The recombinant DNA of the present invention, which contains the HBxAggene can be produced, for example, by cloning a portion of the HBV DNA.To obtain HBV genomic material, Dane particle DNA containing a singlestranded portion is converted to a completely double-stranded DNA byusing the virus' endogenous DNA polymerase.

The circular, double-stranded HBV thus obtained contains two BamHIendonuclease restriction sites. Cleavage with BamHI typically yieldsthree linear products classified by size. The largest is a 3200 basepair (bp) fragment representing the entire HBV genome cleaved at onlyone BamHI site. Fragments containing 1850 bp and 1350 bp are producedwhen HBV is cleaved at both BamHI sites.

Analysis of the HBV nucleotide sequence of Galibert et al. (1979),Nature, 281:646-650, reveals that the 1850 bp HBV BamHI fragmentincludes the gene coding for HBxAg. However, all three fragments haveBamHI complementary termini and may therefore be inserted into the BamHIsite of a cloning plasmid.

The plasmid pBR322 has a single BamHI restriction site that is locatedwithin its tetracycline resistance-conferring gene as can be seen fromexamination of FIG. 3. Cleavage of pBR322 DNA with BamHI yields a singlelinear fragment with termini complementary for each of the three BamHIHBV DNA fragments.

Ligating each of the three BamHI HBV DNA fragments into pBR322 at itsBamHI site with T4 DNA ligase to reform and circularize the plasmidsresulted in three unique recombinant plasmid DNAs. The recombinantcloning plasmids are circular DNAs, and are designated: AM7, containingpBR322 DNA with the 1350 bp BamHI HBV DNA fragment inserted at pBR322'sBamHI site; AM6, containing pBR322 DNA with the entire HBV genomeinserted at pBR322's BamHI site; and AM1, containing pBR322 DNA with the1850 bp BamHI HBV DNA fragment including the HBxAg gene inserted atpBR322's BamHI site.

When the BamHI HBV DNA fragments are ligated into the pBR322 BamHI sitewith T4 DNA ligase, the recombinant plasmids so generated no longer havethe ability to confer tetracycline resistance. Ampicillin resistant andtetracycline sensitive transformants were thereby selected from amongcolonies of the Escherichia coli (E. coli) strain HB101 transformed withthe above ligation products.

Three drug selected strains of E. coli HB101 transformed withrecombinant plasmids AM1, AM6 and AM7 are designated ECAM1, ECAM6 andECAM7, respectively. The above plasmids were prepared in relativelylarge quantities by growing the above mentioned transformants in anappropriate medium such as LB broth, as described hereinafter.

From the above plasmids AM1 and AM6, a gene fragment containing thewhole or part of the HBxAg gene base sequence may be excised by usingappropriate restriction enzymes. For instance, when plasmids AM1 and AM6were digested with the restriction enzyme BamHI, an 1850 bp DNA fragmentincluding the gene coding for HBxAg was isolated from the reactionproducts. By growing the recombinant plasmid-transformed E. coli HB101strains ECAM6 or ECAM1, a relatively large amount of the DNA sequencecoding for HBxAg can be obtained.

From the HBxAg gene for which the DNA base sequence and the locus on theHBV genome have been determined, it is evident that no introns existtherewithin. This means that the gene can be transcribed as it is tolead to phenotypic expression as a messenger RNA in animal cells as wellas in bacterial cells.

2. Expression Vectors

Introduction of the HBxAg gene with an appropriate expression systeminto an animal cell, for example, an African Green Monkey kidney cell bythe method of Ganem et al. (1976), J. Mol. Biol., 101:57-83, can lead toHBxAg synthesis within said cell. Furthermore, cultivation of thosemonkey kidney cells containing the HBxAg gene with an appropriateexpression vector enables low-cost mass production of HBxAg.

Advantageously, a recombinant DNA capable of expressing the HBxAg genewas constructed by inserting, without shifting its reading frame, thecomplete HBxAg gene, or a fragment thereof, into the Simian Virus 40(SV40) downstream from the SV40 late region promoter. A vector forexpression using the SV40 late region promoter was produced in largequantities in the following manner.

SV40 DNA and pBR322 DNA were subjected to BamHI and EcoRI doublerestriction enzyme digestion. The larger SV40 and pBR322 fragments wereligated using T4 DNA ligase. The ligation product was subjected to BamHIdigestion forming a linear DNA with BamHI cohesive termini. Ligation ofthis SV40/pBR322 DNA with the 1850 bp BamHI HBV DNA resulted in acircular recombinant plasmid denominated SVAM191.

The recombinant DNA SVAM191 contains an intact E. coli ampicillinresistance gene. E. coli HB101 transformed with SVAM191 are ampicillinresistant and tetracycline sensitive, and thus may be selected for basedon drug sensitivity. E. coli HB101 transformed with SVAM191 aredesignated EC-SVAM191 , and may be grown in an appropriate medium, suchas LB broth containing ampicillin, and a relatively large amount of saidplasmid may thereby be obtained.

3. Cell Culture

The transformation of a host cell with the thus-obtained recombinant DNAplasmids AM1, AM6 and SVAM191 can be conducted by known methods [Cohenet al., Proc. Natl. Acad. Sci. USA, 69:2110-2114 (1972)] or amodification thereof. The host cells include, among others, suchmicroorganisms as Escherichia coli (E. coli), Bacillus subtilis andyeasts, and preferably are an Escherichia coli strain such as thestrains denominated 294 (ATCC 31446), W3110 (ATCC 27325) or RR1 (ATCC31447). E. coli strain HB101 (ATCC 33694) is a particularly preferredhost cell line.

Isolating a cell strain carrying the HBxAg gene-containing novelrecombinant plasmid DNA may be accomplished by known methods including,for instance, the following technique.

Dane particle DNA which is only partially double-stranded may beradioactively labeled by filling in the single-stranded portion with ³H-containing dNTPs using the endogenous DNA polymerase reaction.Robinson (1975), Am. J. Med. Sci., 270:151-159. Thereafter, using thelabeled product as a probe, a positive clone can be picked out fromamong the already-obtained drug-selected transformants by the knownSouthern Blot Hybridization method [Southern (1975), J. Mol. Biol.,98:503], or by the known colony hybridization method [Grunstein etal.(1975), Proc. Natl. Acad. Sci. USA, 72:3961-3965].

Host cells transformed and isolated in this manner are grown in a knownmedium. The medium may be, for instance, LB broth, Penassay broth, or M9medium containing glucose and Casamino Acids [Miller, Experiments inMolecular Genetics, 431-433 (Cold Spring Harbor Laboratory, New York,1972)].

The cultivation is generally carried out at 15-43 degrees C., preferablyat 28-40 degrees C., for 2-24 hours, preferably for 4-16 hours, ifnecessary with aeration and/or agitation.

After the cultivation, the bacterial cells are collected, the cells aresuspended in a buffer and lysed, as by lysozyme, freeze-thawing orultrasonic treatment. The gene coding for HBxAg may be isolated by amethod generally known for DNA purification from the centrifugationsupernatant obtained following cell lysis.

To produce a vector for expressing HBxAg in eucaryotic cells, a portionof the SVAM191 DNA, including all the DNA of pBR322 origin, was removed.This was accomplished by subjecting SVAM191 to HaeII restriction enzymedigestion. When the larger of the HaeII SVAM191 digestion products wascircularized, a vector capable of expressing HBxAg in animal cells,designated SVHBV-3, was formed.

A mammalian cell host may be transfected with SVHBV-3 by known methodsto provide an expression system. For instance, SVHBV-3 when cleaved withthe restriction enzyme HindIII may be inserted into COS-7 cells (ATCCCRL 1651). [See, Gluzman, (1981), Cell, 23:175-182; and Siddiqui (1983),Mol. and Cell Biology, 3:143-146.] SVHBV-3 may also be inserted inBovine Papilloma Virus and thereby used to transfect murine cell lines.

More preferably, SVHBV-3 is used along with SV40tsA₂₃₉ helper virus totransfect the African Green Monkey kidney cell line BSC-1 (ATCC CCL 26).When so transfected, BSC-1 cells produced a polypeptide containing HBxAgantigenic determinants.

It should be understood that tne nucleotide sequence or gene fragmentinserted at the selected restriction site of the cloning or expressionvehicle may include nucleotides that are not part of the actual gene forthe desired protein or may include only a fragment of that gene. Thus,because of the known redundancies in the genetic code, the inserted geneneed not be identical to the HBxAg gene described herein, but need onlycode for HBxAg. In addition, a DNA sequence that codes for HBxAg mayinclude additional bases at either or both of the 3'- or 5'-ends of thesequence that codes for HBxAg, so long as the reading frame isunchanged. Put differently, whatever DNA sequence is inserted, thepresent invention requires only that the transformed or transfected hostproduce either a DNA coding for HBxAg, the protein HBxAg, or apolypeptide that includes a substantial portion of the HBxAg protein,respectively.

4. Polypeptides, Antigens and Antibodies

As described hereinafter, the expression of HBxAg can be detected usingantibodies induced by synthetic polypeptides whose amino acid residuesequence corresponds to the amino acid residue sequence of an antigenicdeterminant of HBxAg. The polypeptides of tnis invention typicallycontain about 6 to about 40 amino acid residues. More preferably, thosepolypeptides contain about 10 to about 20 amino acid residues.

The amino acid residue sequence of synthetic polypeptides designated 99,and 100 and 142 shown below and in FIG. 6 were determined from the HBVnucleotide sequence as published by Galibert et al., supra.

Polypeptide 99:Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp-Cys,

Polypeptide 100:Cys-Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val, and

Polypeptide 142: Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala.

wherein each of the amino acid residue sequences is shown in thedirection from left to right and in the direction from amino-terminus tocarboxy-terminus.

It is noted that polypeptides corresponding in amino acid residuesequence to the sequences of polypeptides 99 and 100 except for theabsence of the carboxy- and amino-terminal cysteine residues ofpolypeptides 99 and 100, respectively, are also useful herein and areconsidered a part of this invention. Such polypeptides designatedpolypeptides 99b and 100b are useful as immunogens when bound to acarrier through their amino- or carboxy-terminal residues and may alsobe used in immunoreaction blocking studies similar to those shown inportions of FIGS. 7, 8, and 10 and discussed hereafter.

The amino acid residue sequences of polypeptides 99b and 100b are shownin the formulae below from left to right and in the direction frimamino-terminus to carboxy-terminus:

Polypeptide 99b:Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp,

Polypeptide 100b:Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val.

a. X Protein in Transfected Cells

Rabbits immunized with polypeptides 99, 100 or 142 linked to a keyholelimpet hemocyanin (KLH) carrier as a conjugate produced antibodiesanti-99, anti-100, and anti-142, respectively) that immunoreacted withthe HBxAg polypeptide expressed in BSC-1 cells transfected with SVHBV-3,thereby demonstrating for the first time the expression of an HBxAgpolypeptide in those or any cells. Those anti-polypeptide antibodies didnot immunoreact with any polypeptide found in non-transfected BSC-1cells; i.e., cells infected with wild type (wt) SV40.

In addition, the immunoreactivity of the anti-polypeptide antibodieswhich expressed the HBxAg polypeptide was inhibited or blocked bypre-incubation of the antibodies with their complementary(corresponding) polypeptide; i.e., the polypeptide immunogen to whichthey were raised. That blocking indicates that the antibodiesspecifically recognized an antigenic determinant of the polypeptidepredicted to be HBxAg, and that the synthetic polypeptides correspondedin amino acid residue sequence to antigen determinants of the predictedHBxAg protein. These blocking results were obtained using the WesternBlot technique (Materials and Methods) followed by the location of boundantibodies with ¹²⁵ I-labeled Staphylococcus aureus protein A, followedby autoradiographic development of the assay.

These results are shown in FIG. 10 for antisera to polypeptides 99 and142. Lane 1 of FIG. 10 shows the immunoreaction of anti-99 with theSVHBV-3 infected cell lysate, while lane 2 shows blockage of thatimmunoreaction by pre-incubation of the antiserum with polypeptide 99.Similarly, anti-142 immunoreacted with a cell lysate protein in lane 3,and that immunoreaction was blocked by pre-incubation of that antiserumwith polypeptide 142. Lanes 5 and 6 show that no immunoreaction tookplace with lysates from control cells.

The polypeptide expression product of SVHBV-3 transfected cells that wasspecifically identified by antisera to polypeptides 99 and 142, as shownin FIG. 10, had a molecular weight of about 24,000 daltons. As discussedbelow, a polypetpide having a molecular weight of about 28,000 daltonshas been identified in lysates from the human hepatoma-derived cell linePLC/PRF/5. It is believed that the molecular weight difference in thepolypeptides (proteins) expressed by the two cell lines stems from thefact that the polypeptides expressed result from fusion of the X proteinwith other proteins or polypeptides.

Thus, as already noted, SVHBV-3 lacks a portion of the the completeX-encoding genome. FIG. 6 shows that the portion of the X gene includedin SVBHV-3 excludes codons for the first twenty-two amino acid residuesof the putative X protein including the methionine initiation codon ATG.

It was therefore predicted that the polypeptide expressed by cellstransfected by SVHBV-3 would be a fusion product with the SV40structural protein VP2 sequences present in the vector. The predictedsize of the fusion protein (polypeptide) was predicted to be 24,500daltons. The finding of an expressed polypeptide having a molecularweight of about 24,000 daltons thereby conforms to that prediction. The28,000 dalton fusion protein (polypeptide) is discussed hereinafter.

b. X Protein Expressed in Hepatoma Cells

Expression of BHxAg is also believed to be a function of some HBVdisease states. For example, antibodies to polypeptide 99 (anti-99)specifically recognized a 28,000 dalton protein expressed in the humanhepatoma-derived cell line PLC/PRF/5 [ATCC CRL 8024). Normal rabbitserum did not recognize this protein and anti-99 recognition was blockedby pre-incubation with polypeptide 99. This cell line is known tocontain integrated HBV DNA. Chakraborty et al. (1980), Nature,286:531-533; Edman et al. (1980), Nature, 286:535-538; and Marion et al.(1980), Virol, 33:795-806. HBsAg has been detected in this hepatoma cellline, [McNab et al. (1976), Cancer, 34:509-515; and Aden et al. (1979),Nature, 282:615-616], while all standard assays for HBcAg and HBeAg havebeen negative. The cell line denominated HepG2 (ATCC CCL 23) is also ahuman hepatom a cell line, but does not contain integrated HBV DNAsequences. Aden et al., supra.

These results were obtained by radiolabeling the PLC/PRF/5 cell proteinsfollowed by repeated immunoprecipitations using anti-99 andStaphylococcus aureus protein A and then electrophoresis of therecovered precipitate. The 28,000 dalton protein was then identified byautoradiography. This procedure is also discussed in the Materials andMethods section.

Results from a similar study using lysates from both PLC/PRF/5 and HepG2cells are shown in FIG. 7. FIG. 7 demonstrates that an X-specificprotein at 28,000 daltons was detected in PLC/PRF/5 cells (lane 5). Theimmunoreactivity was lost when antibody was preincubated withpolypeptide 99 (lane 6), but not with a non-related polypeptide (lane7). The preimmune serum control did not immunoreact (lane 8). NoX-specific reactivity was detected in the control HepG2 lysates (lanes1-4). These results establish that an X gene product is being expressedin a human hepatoma cell line containing integrated HBV DNA sequences.

c. X Protein Expression in Liver Cells

In addition, four liver samples from human and chimpanzee, either withor without a history of HBV infection, were examined in a Western blotassay for the presence of an X related protein using anti-X polypeptideantibodies. The results of this examination are shown in FIG. 8. Onehuman sample (H) was from a chronically HBV infected hepatoma liver andthe other from an acute phase HBV infection (H). An uninfectedchimpanzee (C) and one chronically infected with HBV (C) were both usedto obtain liver cell lysates that were reacted against anti-Xpolypeptide antibodies.

Anti-99 antibodies reacted against both a 45,000 and 28,000 (left-marginarrow, right-margin p28) dalton protein. Both reactivities were blockedwhen these antibodies were preincubated with polypeptide 99. Whenanti-142 antibodies were reacted against these same cell lystates,specific bands at 40,000 (p40), 28,000 (p28) and 17,000 (p17) weredetected. The reactivities against these proteins are consideredspecific because they are removed when anti-142 antibodies arepreincubated with polypeptide 142.

The control sample, an uninfected chimpanzee liver (lane 3 of allpanels), exhibited the about 45,000 dalton protein (p45) with anti-99antibodies and both p40 and p17 with anti-142 antibodies. Protein p28was expressed in HBV infected tissues only (lanes cells (lane 5). Theimmunoreactivity was lost when antibody was preincubated withpolypeptide 99 (lane 6), but not with a non-related polypeptide (lane7). The preimmune serum control did not immunoreact (lane 8). NoX-specific reactivity was detected in the control HepG2 lysates (lanes1-4). These results establish that an X gene product is being expressedin a human hepatoma cell line containing integrated HBV DNA sequences.

The presence of a 45,000 dalton protein was also observed in arelatively small amount in the lysates from PLC/PRF/5 cells shown inFIG. 7. The immunoreaction between anti-99 antibodies and the 45,000dalton protein was also blocked by pre-incubation of the antibodies withthe immunizing polypeptide 99. This is shown in lane 6 of FIG. 7.

Normal human liver cell extracts (HBV seronegative) were found toexpress only the 45,000 dalton protein in the Western Blot assay usinganti-99. In addition, control assays run using commercially availableantibodies raised to HBs, HBc and HBe (Abbott Laboratories, NorthChicago, IL) failed to identify either protein located by the antibodiesof this invention. This evidence again suggests that the 45,000 daltonprotein has antigenic determinants homologous with HBxAg, but that the28,000 dalton protein recognized by anti-99 is specific for a HBVdisease state, and is different from HBsAg, HBeAg and HBcAg.

d. X Gene in Liver Cells

The expression of p28 in PLC/PRF/5 cells was from HBV DNA integratedinto the host DNA. It was of interest to determine if this were the onlyway in which X protein could be expressed.

Therefore, those chimpanzee and human liver DNAs that exhibited Xprotein were probed with HBV DNA (FIG. 9). Total DNA was isolated fromthe liver tissues represented in FIG. 8, and were analyzed for thepresence of HBV-DNA sequences. The DNA from the 28,000 daltonprotein-containing chimpanzee revealed homologous bands when cleavedwith BamHI, EcoRI or when undigested (FIG. 9, lanes 1, 2 and 3respectively). BamHI cleaves circular HBV DNA into two fragments [1850and 1350 base pairs (bp)], the band at 5.8 kilobases (Kb) (lane 1)represents a larger than genome size integrated sequence. Since no bandswere seen below the high molecular weight DNA band (lane 3) it isconcluded that only integrated sequences of HBV are present in this DNA.

In addition, DNA from an HBcAg-positive human liver was prepared, andwas probed with HBV DNA. Restriction enzyme cleavage with HindIII,BamHI, EcoRI or undigested DNA (FIG. 9, lanes 7-10, respectively)revealed homologous sequences either equal to or smaller than genomesize, thereby eliminating any evidence that these human DNA samplescontain integrated sequences. Chimp and human liver tissues lacking the28,000 dalton protein were also negative for HBV DNA sequences (FIG. 9,lanes 4-6 and 11-14, respectively). The data shown in FIG. 9 confirmthat X protein is expressed in HBV infected tissues regardless of thestate of the HBV genome, and therefore eliminate a prerequisite of HBVDNA integration for X expression.

e. Summary of Results

The results described hereinbefore demonstrate the existence of apreviously unidentified viral-encoded protein in HBV infected human andchimpanzee liver tissues. This 28,000 dalton protein (p28) is encoded bythe HBV genome whether it exists free in an extrachromosomal form or isfound integrated into the cellular DNA.

The 28,000 dalton protein (p28) is not the product of the X regionalone, but contains additional HBV sequences. The predicted size of Xprotein, based on the sequence reported by Galibert et al. supra, isabout 17,000 daltons (FIG. 6)). However, the molecular weight of the Xprotein detected by anti-X peptide antibodies in PLC/PRF/5 cells, aswell as in chimpanzee and human HBV-infected tissues is 28,000 daltons.The discrepancy in size is not due to a fusion of the X gene withcellular sequences since the lack of integrated HBV DNA in human tissuesexpressing p28 (FIG. 9, lanes 7-10) indicates that the X protein (p28)is translated solely from the HBV genome.

The increased size of the X protein can be explained if the protein iseither an HBsAg-X or an X-HBcAg fusion. The probability of such a fusionis suggested by the proximty of these HBV genes to one another on thegenome.

An attempt to determine the existence of such a fusion on the RNA levelwas unsuccessful, due to the fact that the transcripts obtained in thePLC/PRF/5 cells, which were found to contain X sequences, were alsoexhibiting either surface or core sequences as well as X (data notshown). Similar results were reported by Gough (1983), J. Mol. Biol.,165:683-699. Evidence for an X-core fusion comes from the DNA sequenceof another member of the Hepadnavirus family, the duck hepatitis B virus(DHBV), where both genes are translated as a single protein [Mandart, etal. (1984), J. Virol., 49:782-792] and from work by Feitelson et al.(1982), J. Virol., 43:687-696.

The fact that the X region of HBV is expressed has been established.However the function of this gene product remains unknown. A proteinmolecule, which has been sugested to be the translation product of the Xregion, is found in association with the HBV genome, or morespecifically, is covalently linked to the 5' nick of the L strandGerlich et al. (1980), Cell, 21:801-809. The idea of X proteinrepresenting a DNA binding protein was eliminated when DNAase treatmentof PLC/PRF/5 cells or HBV infected tissues failed to remove or alter p28activity (data not shown).

Presently, little is known concerning the biolgy of HBV infection, orthe correlation made between HBV infection and the subsequent onset ofhepatocellular carcinoma. To this end, any additional marker for HBVinfection or for the presence of HBV sequences is of major importance.Human sera are presently being examined for the existence of anti-Xantibodies and the X protein.

The term "correspond" in its various grammatical forms is used hereinand in the claims in relation to polypeptide sequences to mean thepolypeptide sequence described plus or minus up to three amino acidresidues at either or both of the amino- and carboxy-termini andcontaining only conservative substitutions in particular amino acidresidues along the polypeptide sequence.

The term "conservative substitution" as used above is meant to denotethat one amino acid residue has been replaced by another, biologicallysimilar residue. Examples of conservative substitutions include thesubstitution of one hydrophobic residue such as Ile, Val, Leu or Met foranother, or the substitution of one polar residue for another such asbetween Arg and Lys, between Glu and Asp or between Gln and Asn, and thelike.

In some instances, the replacement of an ionic residue by an oppositelycharged ionic residue such as Asp by Lys has been termed conservative inthe art in that those ionic groups are thought to merely providesolubility assistance. In general, however, since the replacementsdiscussed herein are on relatively short synthetic polypeptide antigens,as compared to a whole protein, replacement of an ionic residue byanother ionic residue of opposite charge is considered herein to be a"radical replacement", as are replacements between nonionic and ionicresidues, and bulky residues such as Phe, Tyr or Trp and less bulkyresidues such as Gly, Ile and Val.

The terms "nonionic" and "ionic" residues are used herein in their usualsense to designate those amino acid residues that normally either bearno charge or normally bear a charge, respectively, at physiological pHvalues. Exemplary nonionic residues include Thr and Gln, while exemplaryionic residues include Arg and Asp.

The word "antigen" has been used historically to designate an entitythat is bound by an antibody, and also to designate the entity thatinduces the production of the antibody. More current usage limits themeaning of antigen to that entity bound by an antibody, while the word"immunogen" is used for the entity that induces antibody production.

In some instances, the antigen and immunogen are the same entity aswhere a synthetic polypeptide is utilized to induce production ofantibodies that bind to the polypeptide. However, the same polypeptides(99, 100 or 142) also induce antibodies that bind to a whole proteinsuch as HBxAg, in which case the polypeptide is both immunogen andantigen, while the HBxAg is an antigen. Where an entity discussed hereinis both immunogenic and antigenic, it will generally be termed anantigen.

5. Assay Systems and Methods

The above results indicate that SVHBV-3 may be used as an expressionvector. It provides expression control elements for foreign DNAsequences inserted in reading frame downstream from the HBxAg gene.

The results also indicate that synthetic polypeptides 99, 100 and 142,and antipeptide antibodies thereto may be used as part of an assaysystem and method for detecting the presence of HBxAg in a body sampleto be assayed such as hepotoma cells or liver cells from an animal hostsuspected of having HBxAg, to detect successful transfection withSVHBV-3, and to monitor expression when SVHBV-3 is used as an expressionvector.

Such a system includes at least a first reagent containing receptormolecules that include an antibody combining site such as antibodies,substantially whole antibodies or the well known Fab and F(ab')₂antibody portions, and are capable of immunoreacting with a syntheticpolypeptide of this invention; i.e., raised to the syntheticpolypeptides of this invention. Indicating means, such as a fluorescentdye like fluorescein or rhodamine, a radioactive element like iodine-125or carbon-14, or an enzyme-linked antibody raised to the first reagent'sreceptors (e.g. peroxidase-linked goat anti-rabbit IgG), to signalimmunoreaction of the receptors of the first reagent with the expressionspecific HBxAg are also provided. The indicating means may be initiallybound or free from the first reagent. Typical uses of such a HBxAg assayreagent system include enzyme-linked immunosorbent assays (ELISA),radioimmunoassays and immunofluorescent assays (IF).

Diagnostic assays for determining the presence of HBxAg in a body sampleutilizing the polypeptides of this invention and their anti-polypeptideantibodies have already been broadly described. In a commercialembodiment of such a diagnostic assay, a diagnostic assay system iscontemplated.

One such system includes provision of at least one container having ananti-polypeptide receptor such as anti-99 or anti-142 as the firstreagent. A second container having a quantity of the polypeptide towhich the anti-polypeptide antibody was raised, e.g. polypeptide 99 orpolypeptide 142, may also be supplied to provide a second reagent.Additional containers having the before-described indicating means, oneor more buffers, and the like as are well known may also be provided tothe diagnostic assay system.

A method for assaying for the presence of a detectable amount of HBxAghas also been broadly described hereinbefore. Briefly, that methodincludes admixing proteins from a body sample to be assayed such ashepatoma or liver cells with receptors that include an antibodycombining site capable of immunoreacting with a synthetic polypeptide ofthis invention and with HBxAg, such as anti-99 or anti-142, or an Fab orF(ab') portion thereof, in the presence of an indicating means such as¹²⁵ I-labeled Staphylococcus aureus (S. aureus) protein A or an enzymelabel, like horseradish peroxidase linked to one of the above receptorsfor signalling an imunoreaction between HBxAg and the receptors. Theadmixture is maintained for a time period sufficient for the indicatingmeans to signal that an immunoreaction has occured, and the presence ofthe signal is ascertained as by an autoradiogram. The body sample, orthe proteins therefrom, is preferably adsorbed or otherwise affixed(bound) to a solid matrix such as a nitrocellulose sheet or glass slideprior to being admixed with the receptors.

Where the indicating means is a separate molecule such as radiolabeledS. aureus protein A, the bound body sample and receptor molecules areadmixed first, in the absence of the indicating means, and the admixtureis maintained for a time period sufficient to form a boundimmunoreaction product. The bound immunoreaction product is then rinsed.The separate molecule indicating means is then admixed with the boundimmunoreaction product, and that admixture is maintained for a timeperiod sufficient for a bound, second reaction product to form. Thebound second reaction product is then rinsed, and the presence of asignal from the indicating means is determined.

Where the indicating means is a portion of the receptors of thisinvention, those labeled receptors are admixed with the bound bodysample to be assayed, and the admixture is maintained for a time periodsufficient for an immunoreaction product to form. The boundimmunoreaction product is then rinsed, and the presence of a signal fromthe indicating means is determined.

The beforementioned second reagent, e.g., polypeptides 99 and 142, maybe used as a control reagent in an immunoreaction blocking study toverify that specific, rather than non-specific, immunobinding hasoccurred. Such blocking studies are illustrated in FIGS. 7, 8 and 10.

To use SVHBV-3 as an expression vector, foreign DNA is inserteddownstream from the HBxAg gene, preferably at its unique BamHIrestriction site, and the new vector so produced is utilized totransfect cells. Expression of HBxAg in a transfected cell ispresumptive evidence of expression of the foreign DNA inserted intoSVHBV-3. Expression of HBxAg detected by the above described HBxAgexpression assay system indicates the espression of foreign DNA insertedinto SVHBV-3.

Thus, examination of cell colonies produced after the attemptedtransfection using the above assay system can be utilized to selectthose colonies in which transfection was successful. For example, cellsfrom colonies resulting from the attempted introduction of the vectorinto eucaryotic cells are harvested, lysed, and the cellular proteinsare extracted. The extracted cellular proteins are thereafter separatedon a SDS-polyacrylamide gel by electrophoresis, and the separatedproteins are blotted onto nitrocellulose. Contacting the blottedproteins with an excess of receptor and label of the before-describedassay system such as ¹²⁵ I-labeled antibodies to synthetic polypeptide99 or polypeptide 142, followed by rinsing to remove thenon-immunoreacted antibodies (receptors) can then be used to prepare anautoradiograph that indicates the presence of the expressed HBxAgportion that is fused to the polypeptide encoded by the foreign gene.

The before described assay methods and systems are particularly usefulfor identifying the X protein or a substantial portion thereof that maybe present in a body sample, particularly a tissue sample such as incells from a liver biopsy. The diagnostic assay and method describedhereinbelow is particularly useful for determining the presence ofantibodies to the X protein that may be present in a body sample such aswhole blood, serum or plasma. A Western blot analysis will be used asexemplary of such a diagnostic method. However, a commercial embodimentthat utilizes the method is an ELISA (enzyme-linked immunosorbant assay)diagnostic system.

Here, an expression product from SVHBV-3 vector-transfected cells suchas the approximately 24,000 dalton protein (polypeptide) expressed inBSC-1 cells discussed in relation to FIG. 10 is preferably utilized asthe antigen, although a preferred polypeptide of this invention such aspolypeptide 99 or polypeptide 142 may also be used as an antigen. Thus,the HBxAg molecule itself, used in substantially purified form asobtained by affinity chromatography as discussed hereinafter, asubstantial portion of the HBxAg molecule such as is expressed bySVHBV-3 transfected BSC-1 cells (the 24,000 dalton polypeptide), or apolypeptide corresponding in amino acid residue sequence to an antigenicdeterminant of HBxAg may be used as the antigen.

The antigen is preferably bound on (adsorbed to) or otherwise affixed toa solid matrix such as the cross-linked dextran available under thetrademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.),agarose, beads of glass, polyvinyl chloride, polystyrene, cross-linkedacrylamide, nitrocellulose or the wells of a microtiter plate to form asolid support.

The antigen, preferably bound to a solid matrix as part of a solidsupport, is admixed with a liquid body sample to be assayed to form asolid-liquid phase admixture. The admixture is maintained for a timeperiod sufficient for anti-X protein (anti-HBxAg) antibodies present inthe body sample to immunoreact with the antigen. The presence of thatimmunoreaction is then determined as with an indicating means to signalthe presence of anti-X protein antibodies in the assayed body sample

For example, in one study, SVHBV-3 vector-transfected BSC-1 cell lysateswere prepared, separated electrophoretically, and were transferred andbound to nitrocellulose sheets as a solid matrix in a mannersubstantially similar to that discussed in relation to FIG. 10 to form asolid phase. Sera diluted 1:50 from six patients with various hepatiticdiseases were then separately admixed with the nitrocellulose-boundtransfected BSC-1 cell proteins to form solid-liquid phase admixtures.Those admixtures were maintained for a time period (2 hours) sufficientfor anti-X antibodies present in the sera to immunoreact with the boundantigen.

After rinsing to separate the solid and liquid phases, the solid phasesresulting from the above steps were separately admixed with liquidsolutions containing 1:200 dilutions of goat anti-human or anti-rabbitantibodies conjugated to horseradish perioxidase as an indicating meansto form second, solid-liquid phase admixtures. The separate, second,solid-liquid phase admixtures were maintained for a time period (1 hour)sufficient for the labeled antibodies to react with human anti-Xantibodies that had immunoreacted with the matrix-bound antigen. Rabbitanti-polypeptide antibodies were used as controls with the goatanti-rabbit antibodies. The resulting solid-liquid phases admixture wereagain separated and rinsed. The resulting solid phases were thendeveloped using solutions containing 4-chloro-1-naphthol as describedfor FIG. 8.

The results of this study indicate that serum from a patient diagnosedas having a heptacellular carcinoma contained antibodies that bound tothe polypeptide expressed by the SVHBV-3 vector in the transfected BSC-1cells. Those antibodies are thus anti-X antibodies, and their presenceestablishes the X gene product as an authentic antigen at some stage ofHBV infection.

Where the approximately 24,000 dalton polypepiide expression productused in the above-described Western blot assay is utilized as theantigen in an ELISA, as discussed hereinbelow, or in other similarassays, that polypeptide is preferably purified and isolated prior tosuch use. That purification and isolation may be achieved by well knowntechniques.

For example, anti-99 or anti-142 antibodies, or the antibody combiningsites thereof, may be prepared as described herein and linked to a solidmatrix such as the agarose and cross-linked agarose derivatives soldunder the trademarks SEPHAROSE 6B, CL6B, 4B and CL4B by Pharmacia FineChemicals or those sold under the trademarks Bio-Gel A-0.5M, A-1.5M, andA-50M by Bio-Rad Laboratories of Richmond CA. The before-describedcross-linked dextran sold under the trademark SEPHADEX, andpolyacrylamide beads sold under the trademarks Bio-Gel P-2, P-30, P-100and P-300 also by Bio-Rad Laboratories are also useful solid matrixes.Use of a matrix comprised of an agarose derivative is discussedillustratively below.

The agarose matrix is typically activated for linking using cyanogenbromide. The activated matrix is then washed and linked to the desiredantibody (receptor) molecules without drying of the activated matrix.The matrix-linked antibody is then washed and is ready for use.Unreacted reactive groups on the matrix can be reacted with an aminesuch as ethanolamine or Tris, if desired, although those reactive groupsdecay quickly.

The affinity sorbant may be used in its loose state, as in a beaker orflask, or it may be confined in a column. Prior to use, it is preferablethat the affinity sorbant be washed in the buffer or other aqueousmedium utilized for purification of cell lysates containing theapproximately 24,000 dalton polypeptide to eliminate non-specificallybound proteins or those antibodies that were unstably linked to thesupport.

An aqueous composition containing the SVHBV-3 vector-transfected celllysate is provided, and is then admixed with the affinity sorbant. Thatadmixture forms a reversible, linked antibody-antigen (receptor-ligand)complex between the linked antibody (receptor) and the approximately24,000 dalton polypeptide (antigen).

The linked receptor-ligand complex is then separated from the remainderof the un-complexed aqueous composition to thereby obtain thepolypeptide antigen in purified form linked to the affinity sorbant.When the admixture takes place in a beaker or flask, this separation canbe made by filtration and washing. When the sorbant is in a column, theseparation may take place by elution of the un-complexed aqueous medium,again, preferably, follwed by a washing step.

When the purified polypeptide antigen is desired free from the affinitysorbant, it can typically be obtained by a variety of procedures. In anyof those procedures, the reversible linked receptor-ligand complex isdissociated into its component parts of matrix-linked receptor andpolypeptide antigen ligand, followed by separating that polypeptideantigen ligand from the linked-receptor to provide a solution of thepurified polypeptide antigen ligand free from the affinity sorbant. Thesolution may be used as such, or it may be concentrated or dried priorto use as desired. In some instances, it may be desirable to desalt thesolution prior to use, as is known.

The dissociation of the reversible complex may be effected in a numberof ways. A 0.2 molar glycine hydrochloride solution at a pH value ofabout 2.5 is typically utilized. Alternatively, the bound polypeptideantigen ligand can be competed away from the linked receptor byadmixture of the reversible complex with an excess of the immunogenicpolypeptide utilized to raise the antibodies, e.g., polypeptide 99 whereanti-99 antibodies are linked to the matrix. Such a competition avoidspossible denaturation of the polypeptide antigen. Separation of thedissociated polypeptide antigen from the affinty sorbant may be obtainedas above.

The preparation of affinity sorbants and their use is broadly old.However, such materials and uses that incorporate the antibody andantigen molecules of this invention have not been heretofore available.A detailed description of affinity sorbants, their methods ofpreparation and use wherein the antigen is linked to the matrix may befound in Antibody as a Tool, Marchalonis and Warr eds., John Wiley &Sons, New York, pages 64-67 and 76-96 (1982).

An exemplary ELISA utilizing the above method uses a solid supportcomprised of a before-described antigen of this invention adsorbed ontoor otherwise affixed to a solid matrix comprised of the wells of atwelve or ninety-six well microtiter plate made of polystyrene orpolyvinyl chloride to form the solid support. Non-specific binding siteson the microtiter well walls are thereafter typically blocked with aprotein such as bovine serum albumin (BSA). Any unbound antigen and BSAare removed from the microtiter well as by rinsing.

A body sample aliquot such as human serum, blood or plasma is admixedwith the above-described antigen-bound solid support to form anadmixture containing solid and liquid phases. The solid-liquid phaseadmixture is maintained for a time period sufficient for anti-X proteinantibodies in the body sample to immunoreact with the antigen, e.g.about 30 minutes to about 2 hours. The solid and liquid phases arethereafter generally separated.

A liquid solution of a second, labeled, indicating means-containing,antibody, antibody combining site or S. aureus protein A that reactswith the first-named antibody is then admixed with the solid phase toform another solid-liquid phase admixture. An exemplary second antibodyis a peroxidase-labeled goat anti-human Ig antibody where thefirst-named antibodies are from a human body sample. Additional, usefulenzyme labels include alkaline phosphase, beta-D-galactosidase andglucose oxidase.

The admixture formed from the solid phase (the solid matrix-boundantigen-antibody immunoreaction product) and the second, labeledantibody solution is maintained (incubated) for a time period (e.g.,about one hour) sufficient to form a reaction product between the boundfirst-named antibody and the indicating means such as a secondimmunoreaction between the two antibodies. The solid and liquid phasesare thereafter separated.

The second antibody described above may also be specific for andimmunoreact with only one of the classes of immunoglobulin (e.g., IgG,IgM, IgE, IgA, or IgD). Such antibodies provide the ability to identifythe immunoglobulin class of anti-X protein antibody present in the bodysample. In addition, the second antibody or antibody combining site maybe specific for and immunoreact with only one of the two types ofimmunoglobulin light chains (e.g., kappa or lambda). These antibodiesprovide the ability to identify the isotype of the immunoglobulinmolecule present in the body sample, and are well known.

A solution containing a substrate for the enzyme label such as hydrogenperoxide for peroxidase and a color-forming dye precursor such aso-phenylenediamine or 4-chloro-1-naphthol, or p-nitrophenyl phosphatefor alkaline phosphatase, is thereafter admixed with the solid phase.The optical density at a preselected wavelength (e.g., 490 or 405nanometers, respectively) may then be determined after a sufficient timeperiod has elapsed (e.g., 60 minutes), and compared to the opticaldensity of a control to determine whether anti-X protein antibodies werepresent in the body sample.

Another embodiment of this invention comprises a diagnostic system inkit form that includes a solid support comprised of a solid matrix suchas a polystyrene twelve-well microtiter strip, and a before-describedantigen of this invention absorbed (bound) or otherwise affixed to thatsolid matrix to form a solid support. This system preferably alsoincludes separately packaged anti-human Ig antibodies having a linkedindicating means such as peroxidase-labeled goat anti-human Igantibodies, and may also include a substrate for the linked indicatingmeans such as hydrogen peroxide and a color forming due precursor suchas o-phenylenediamine, in further, separate packages. Hydrogen peroxideis typically not included in the kit due to its relative instability,and is typically supplied by the end user, although a container ofhydrogen peroxide may also be a component of the diagnostic system.Buffer salts useful in an assay utilizing this system may also beincluded in one or more separate packages in dry or liquid form. Apreferred polypeptide of this invention such as polypeptide 99 may alsobe included in a separate package for use in competitive binding studiesas a control. An assay for the presence of anti-X protein antibodies ina body sample such as serum may be carried out with this diagnosticsystem using the before-described method.

6. Preparation of Polymers

The polypeptides of the present invention may be connected together toform a water-soluble or water-dispersible antigenic polymer comprising aplurality of the polypeptide repeating units. Such a polymer has theadvantage of increased immunological reaction. Where differentpolypeptides are used to make up the polymer, such polymers have theadditional ability to induce antibodies that immunoreact with aplurality of antigenic determinants of HBxAg.

A polymer may be prepared by synthesizing the polypeptides, as discussedhereinafter, to contain two cysteine residues. The two cysteine residuesmay be present at one terminus and within the polypeptide chain, or atboth the amino- and carboxy-termini. A polypeptide containing twocysteines is referred to generally herein as a "diCys" polypeptide,while a polypeptide that contains tw0 terminal cysteine residues isreferred to herein as a "diCys-terminated" polypeptide. Such cysteineresidues within the polypeptide chain or at the polypetpide chaintermini may be present in the HBxAg sequence to which the polypeptidecorresponds, e.g. the central cysteine of polypeptide 142 (the seventhresidue from the carboxy-terminal alanine (Ala)], the carboxy-terminalcysteine (Cys) of polypeptide 99, or terminal cysteine residues may beadded for the purpose of preparing the polymer.

Exemplary of useful diCys polypeptides are those represented by theformulae shown below, written from left to right and in the directionfrom amino-terminus to carboxy-terimus:

Polypeptide 99a:Cys-Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp-Cys,

Polypeptide 100a:Cys-Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val-Cys,and

Polypeptide 142a:R¹ -Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala-R²,

wherein R¹ and R² are each a cysteine residue, with the proviso thatonly one of R¹ and R²¹ is present.

As is shown in FIG. 6, each of polypeptides 99, 100 and 142 contains acysteine residue that is present in the amino acid residue sequence ofthe translated X-genome. It is reiterated that polypeptides having theamino acid residue sequences of polypeptides 99 and 100 except for theterminal cysteine residues are also useful herein in immunoreactionblocking studies and where coupling to a carrier by a means other thanthe MBS reaction, discussed hereinafter, is used.

When one or two terminal cysteine residues are added for the purpose ofpolymer prepration, and the remaining amino acid residue sequencecorresponds to an antigenic determinant of HBxAg, the polypeptiderepeating unit is still considered to correspond to an antigenicdeterminant of HBxAg.

In a typical laboratory preparation, 10 milligrams of the diCyspolypeptide (containing cysteine residues in un-oxidized form) aredissolved in 250 milliliters of 0.1 molar ammonium bicarbonate bufferhaving a pH value of about 8. The dissolved diCys-terminated polypeptideis then air oxidized by stirring the resulting solution gently for aperiod of about 18 hours, or until there is no detectable free mercaptanby the Ellman test. [See Ellman (1959), Arch. Biochem. Biophys.,82:70-77.]

The polymer so prepared contains a plurality of the polypeptides of thisinvention as repeating units. Those polypeptide repeating units arebonded together by oxidized cysteine residues.

Such a polymer containing a plurality of the polypeptides of thisinvention can be represented by the formula, written from left to rightand in the direction from amino-terminus to carboxy-terminus,

    (A).sub.a, (B).sub.b

wherein A and B are different polypeptide repeating units such aspolypeptides 99, 100 and 142. The subscript letters a and b are integerseach having an average value of zero to about 2000 with the proviso thatthe sum of the average values for a and b is at least two, so that thereare at least two polypeptide repeating units present in the polymer.

The sum of the average values of the superscript letters a and b aretypically not greater than about 2000 so that the resulting polymer hasan average molecular weight of about 5,000,000 daltons. In preferredpractice, the average molecular weight of the polymer is about 50,000 toabout 1,000,000 daltons.

The above formula is intended to be a generalized representation of therepeating units of the polymer and of the average number of eachrepeating units that is present. The polymers of this invention thatcontain two different repeating units are typically random copolymers.Thus, the formula shown hereinbefore is not intended to represent theorder in which the polypeptides repeating units are present in thepolymer, e.g. as in a block copolymer.

The above-described water-soluble or water-dispersible polymers of thisinvention are both immunogenic and antigenic, and are thereforedescribed herein as antigenic, as discussed before. Such antigenicpolymers when dispersed in a physiologically tolerable diluent andintroduced as by immunizing injection in an amount of 400 micrograms ofpolymer per rabbit, are capable of inducing the production of antibodiesin New Zealand Red rabbits that immunoreact with HBxAg. Exemplaryphysiologically tolerable diluents are well known in immunology and arediscussed in further detail hereinafter.

7. Coupling of Polypeptides to Protein Carriers

The synthetic polypeptides utilized herein were coupled to keyholelimpet hemocyanin (KLH) using the following well known method. The KLHcarrier was first activated with m-maleimidobenzoyl-N-hydroxysuccinimideester, and was subsequently coupled to the polypeptide through acysteine residue present at the polypeptide amino-terminus (polypeptide100), the carboxy-terminus (polypeptide 99), or using the centralcysteine of polypeptide 142, by a Michael addition reaction as describedin Liu et al. (1979), Biochem., 80, 690.

A polypeptide of this invention may also be coupled to a carrier throughdifferent means, and may be coupled to carriers other than KLH. Forexample, a polypeptide, e.g. polypeptide 99b, may be coupled to atetanus toxoid carrier through free amino groups, using a 0.04 percentglutaraldehyde solution as is well known. See, for example, Klipstein etal. (1983), J. Infect. Disc., 147:318.

Cysteine residues present at the amino- or carboxy-terminii or in thecentral portion of the synthetic polypeptide have been found to beparticularly useful for forming conjugates via disulfide bonds andMichael addition reaction products, but other methods well known in theart for preparing conjugates can also be used. Exemplary additionalbinding (linking) procedures include the use of dialdehydes such asglutaraldehyde (discussed above) and the like, or the use ofcarbodiimide technology as in the use of a water-soluble carbodiimide,e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, to form amide linksbetween the carrier and polypeptide.

Useful carriers are well known in the art and are generally proteinsthemselves. Exemplary of such carriers are keyhole limpet hemocyanin(KLH), edestin, thyroglobulin, albumins such as bovine serum albumin orhuman serum albumin (BSA or HSA, respectively), red blood cells such assheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well aspolyamino acids such as poly(D-lysine:D-glutamic acid), and the like.

As is also well known in the art, it is often beneficial to bind thesynthetic polypeptide to its carrier by means of an intermediate,linking group. As noted before, glutaraldehyde is one such linkinggroup.

However, when cysteine is used, the intermediate linking group ispreferably an m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), alsodiscussed before. MBS is typically first added to the carrier by anester-amide interchange reaction. Thereafter, the above Michael reactioncan be followed, or the MBS addition may be followed by a Michaeladdition of a blocked mercapto group such as thiolacetic acid (CH₃ COSH)across the maleimido-double bond. After cleavage of the acyl blockinggroup, and a disulfide bond is formed between the deblocked linkinggroup mercaptan and the mercaptan of the added cysteine residue of thesynthetic polypeptide.

The choice of carrier is more dependent upon the ultimate intended useof the antigen than upon the determinant portion of the antigen, and isbased upon criteria not particularly involved in the present invention.For example, if an inoculum is to be used in animals, as for theproduction of anti-polypeptide antibodies to be used to assay for thepresence of HBxAg, a carrier that does not generate an untoward reactionin the particular animal should be selected. If an inoculum such as avaccine against HBxAg is to be used in man, then the overriding concernsinvolve the lack of immunochemical or other side reaction of the carrierand/or the resulting antigen, safety and efficacy--the sameconsiderations that apply to any vaccine intended for human use.

8. Immunization Procedures

The inocula used for immunizations contain an effective amount ofpolypeptide, as a polymer of individual polypeptides linked togetherthrough oxidized cysteine residues or as a conjugate of the polypeptidelinked to a carrier. The effective amount of polypeptide per inoculationdepends, among other things, on the species of animal inoculated, thebody weight of the animal and the chosen inoculation regimen as is wellknown. Inocula are typically prepared from the dried, solidpolypeptide-conjugate or polypeptide polymer by suspending thepolypeptide-conugate or polypeptide polymer in water, saline oradjuvant.

These inocula typically contain polypeptide concentrations of about 20micrograms to about 500 milligrams per inoculation. The stated amountsof polypeptide refer to the weight of polypeptide without the weight ofa carrier, when a carrier was used.

The inocula also contain a physiologically tolerable (acceptable)diluent such as water, phosphate-buffered saline, or saline, and furthertypically include an adjuvant as part of the diluent. Adjuvants such ascomplete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) andalum are materials well known in the art, and are available commerciallyfrom several sources.

Inoculum stock solutions are prepared with CFA, IFA or alum as follows:An amount of the synthetic polypeptide-conjugate or polymericpolypeptide sufficient to provide the desired amount of polypeptide perinoculation is dissolved in phosphate-buffered saline (PBS) at a pHvalue of 7.2. Equal volumes of CFA, IFA or alum are then mixed with thepolypeptide solution to provide an inoculum containing polypeptide,water and adjuvant in which the water-to-oil ratio is about 1:1. Themixture is thereafter homogenized to provide the inoculum stocksolution.

Rabbits used herein to raise anti-polypeptide antibodies were injectedsubcutaneously with an inoculum comprising 200 micrograms of apolypeptide conjugate (polypeptide plus carrier) emulsified in completeFreund's adjuvant (CFA); 200 micrograms of polypeptide conjugate,incomplete in Freund's adjuvant (IFA); and 200 micrograms of polypeptideconjugate with 4 milligrams alum injected intraperitoneally on days 0,14 and 21, respectively, of the immunization schedule. Each inoculation(immunization) consisted of four injections of the inoculum. Mice may beimmunized in a similar way using about one-tenth of the above dose perinjection.

Animals are typically bled 4 and 15 weeks after the first injection.Control pre-immune serum was obtained from each animal by bleeding justbefore the initial immunization.

Control inoculum stock solutions can also be prepared with keyholelimpet hemocyanin (KLH), KLH in IFA (incomplete Freund's adjuvant),KLH-alum absorbed, KLH-alum absorbed-pertussis, edestin, thyroglobulin,tetanus toxoid, tetanus toxoid in IFA, cholera toxoid and cholera toxoidin IFA.

Upon injection or other introduction of the antigen or inoculum into thehost animal, the immune system of the animal responds by producing largeamounts of antibody to the antigen. Since the specific antigenicdeterminant of the manufactured antigen; i.e., the antigen formed fromthe synthetic polypeptide linked to the carrier or the polymer,corresponds to the determinant of the natural antigen of interest, thehost animal manufactures antibodies not only to the syntheticpolypeptide antigen, but also to the protein or polypeptide to which thesynthetic polypeptide antigen corresponds; i.e., to HBxAg.

9. Deposits

The materials enumerated below were placed on deposit in the AmericanType Culture Collection on Mar. 8, 1984, and have the accession numbersindicated for each. All designations for cell lines, vectors and thelike that include the letters "ATCC" followed by numbers and/or lettersand numbers refer to materials deposited with the above American TypeCulture Collection 12301 Parklawn Drive, Rockville, MD 20852.

    ______________________________________                                                         ATTC                                                         Material         Accession Number                                             ______________________________________                                        mixture of virus SVHBV-3                                                                       VR 2084                                                      and SV40 tsA.sub.239 helper                                                   virus.                                                                        Vector DNA                                                                    SVHBV-3          40102                                                        AM6              40101                                                        SVAM191          40103                                                        E. coli                                                                       EC-AM6           39630                                                        EC-AM1           39629                                                        EC-SVAM191       39631                                                        ______________________________________                                    

In addition to the above materials deposited with the ATCC by thepresent inventors, materials prepared by others and used herein havealso been deposited with the ATCC. A list of those materials is providedbelow:

    ______________________________________                                                           ATTC                                                       Material           Accession Number                                           ______________________________________                                        E. coli                                                                       W3110              27325                                                      294                31446                                                      RR1                31447                                                      HB101              33694                                                      Mammalian Cells                                                               HepG2              CCL 23                                                     BSC-1              CCL 26                                                     COS-7              CRL 1651                                                   PLC/PRF/5          CRL 8024                                                   Vector                                                                        pBR322             37017                                                      ______________________________________                                    

B. Materials and Methods 1. HBV DNA Isolation and Preparation

Region XHBV DNA was isolated from Dane particles, subtype adw, from theplasma of an HBsAg-positive human donor [National Institutes of Health#737139) following the procedure of Robinson (Am. J. Med. Sci., (1975)270:151-159]. Briefly, 1.0 ml of plasma was diluted to 3.0 ml with TNbuffer which contained 0.001 molar (M) Tris-HCl (pH 7.4) and 0.5M HCl.The diluted plasma was centrifuged at 10,000xg for ten minutes to removelarge debris, and then layered over 2.5 milliliters (ml) of 30 percent(W/V) sucrose containing TN, 0.001 MEDTA [0.1 percent 2-mercaptoethanoland 1 milligram/milliliter (mg/ml) bovine serum albumin (BSA) that hadbeen previously centrifuged at 10,000xg for 10 minutes to removeprecipitated BSA]. After centrifuging for 4 hours at 50,000 rpm, 4° C.in a Spinco SW-65 rotor (Beckman Instruments, Inc.), the supernatant wascarefully removed and the pellet was resuspended in 50 microliters of TNbuffer containing 1 percent Nonidet P-40 (polyoxyethylene (9) octylphenyl ether; Sigma Chemical Co., St. Louis, MO) and 0.1 percent2-mercaptoethanol.

The single stranded portion of the HBV DNA isolated above was madedouble-stranded by adding 25 microliters of mix E [0.2M Tris-HCl (pH7.4), 0.08M MgCl₂, 0.24 NH₄ Cl, 1.0 millimolar (mM) each of daTP, dTTP,dGTP, and dCTP] to the 50 microliters of resuspend HBV DNA, followed byincubating at 37° C. for 3 hours. Robinson (1975), Am. J. Med. Sci.,270:151-159.

Radioactively labeled HBV DNA to be used as a genomic probe was madeusing the above filling-in procedure by substituting 0.25 microliterseach of 3HdCTP and 3HdGTP (both 21 curies per mM) for dCTP and dGTPrespectively.

2. HBxAg Cloning

A portion of the HBV genome containing the DNA sequence coding for HBxAgwas cloned using the plasmid pBR322 introduced into E. coli.Double-stranded, unlabeled HBV DNA isolated above was digested with therestriction endonuclease BamHI [50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10mM MgCl, 1 mM dithothreitol]. The reaction mixture was electrophoresedin a 0.6% agarose horizontal slab gel at 100 amperes for 2 hours (0.04Tris-Acetate, 0.002M EDTA).

Staining with 1 percent ethidium bromide revealed three HBV DNAfragments, indicating the presence of two BamHI restriction sites in thegenome. A 3200 base pair (bp) fragment represented the entire HBV genomein linear form as a result of being cut at only one of the BamHI sites.The 1850 bp and 1350 bp fragments represented the genome cut into twofragments as a result of complete BamHI digestion.

Copies of the three BamHI HBV DNA restriction fragments isolated abovewere obtained by cloning in the plasmid pBR322 (ATCC 37017) [Sutcliffe(1978), P. Natl. Acad. Sci., USA, 75:3737-3791]. To insert the HBV DNABamHI fragments into pBR322, the plasmid was linearized by digestingwith BamHI [50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mMdithiothreitol] to form termini complimentary to those of the HBVfragments. The three HBV DNA fragments and the pBR322 fragment weremixed, and were subjected to DNA ligation with T4 DNA ligase [66 mMTris-HCl (pH 7.6), 66 mM MgCl₂, 10 mM dithiotheitol and 0.4 mM ATP]. Theligation reaction product obtained in the above reaction is a circularDNA derived from the linearized pBR322 and HBV fragments joining attheir complimentary termini.

The circular recombinant plasmids were introduced into E. coli strainHB101 (ATCC 33694; provided by Dr. Dean Hunter of the National CancerInstitute, NIH, Bethesda, MD) following the procedure of Cohen et al.(1972), Proc. Natl. Acad. Sci. USA, 69:2110-2114. Briefly, an E. coliHB101 culture was incubated in a solution of 0.03M CaCl₂ at zero °C. for20 minutes. About 5×10⁹ bacterial cells were added to 0.3 ml of the samesolution to which was added 100 nanograms (ng) of recombinant plasmids.This transformation reaction mixture was incubated at zero °C. for 60minutes.

The plasmid pBR322 contains ampicillin and tetracyclineresistance-conferring genes. Drug sensitive bacterial cells transformedwith this plasmid exhibit ampicillin and tetracycline resistance. Sincethe pBR322 DNA has only one BamHI cleavage site that is on thetetracycline resistance gene, insertion of the HBV fragments at theBamHI site disrupts this gene. Drug sensitive E. coli HB101 transformedwith a plasmid derived from pBR322 with a HBV DNA fragment inserted atthe BamHI site (cloning vector) therefore exhibit ampicillin resistanceand tetracycline sensitivity.

Transformed E. coli; i.e., those containing the plasmid ampicillinresistance conferring gene, were selected by plating the transformationreaction mixture onto an LB broth agar medium [a growth mediumcontaining, per liter thereof, 10 g Bactoagar, 10 g Bactotryptone, 5 gBacto yeast extract (each from Difco Labs, Detroit, MI) and 5 g NaCl]containing 50 micrograms per ml of ampicillin. The plated E. coli wereincubated at 37° C. for 2 days.

From among the colonies that exhibited ampicillin resistance aftertransformation by the above procedure, tetracycline-sensitive colonieswere selected by plating the colonies onto an LB broth, above,containing 50 micrograms per ml of tetracycline, instead of ampicillin.Those plated colonies were also incubated at 37° C. for 2 days. E. coliHB101 strains containing pB322 with HBV DNA fragments inserted at theBamHI cleavage site in the tetracycline resistance gene were thusobtained.

3. Plasmid DNA Extraction

The E. coli HB101 cells containing the recombinant DNA as isolated abovewere grown in the LB broth medium containing 20 micrograms/mlampicillin, with the addition of chloramphenicol to the concentration of170 micrograms/ml in the logarithmic growth phase. The cultivation wascontinued for several hours for amplifying the plasmid DNA.

The cells were then lysed by admixing 5 ml of a 25 percent sucrose in 50mM Tris-HCl (pH 8.0) solution to 500 ml of cells. After incubation for10 minutes at zero °C., 1 ml of a 1 percent lysozyme in 0.25M Tris-HCl(pH 7.5) solution was admixed. After incubation for 10 minutes atzero°C., 2 ml of 0.25M EDTA (pH 8.0) was gently admixed. Afterincubation for 10 minutes at zero °C. 8 ml of 1 percent Triton X-100[polyoxyethylene (9) octyl phenyl ether] in 0.15M Tris-HCl (pH 8.0),0.2M EDTA (pH 8.0) was admixed and again incubated for 10 minutes atzero °C.

The resulting lysate was centrifuged at 30,000 rpm in a Spinco SW-65rotor (Beckman Instruments) to remove denatured protein and cell debris.DNA was precipitated from the cleared lysate by admixing 1/10 volume of2.5 sodium acetate and 2.5 volumes of 99 percent ethanol, and incubatingat -20° C. for 30 minutes. The DNA precipitate was pelleted bycentrifugation at 10,000xg for 30 minutes.

Deproteinization was performed twice with 1 ml of phenol saturated with0.01M Tris-HCl (pH 7.6), 0.1M NaCl and 0.0012 MEDTA. Landers et al.(1977), J. Virol., 23:368-376. The aqueous layer from the aboveprocedure was taken, and the DNA precipitated therefrom by adding 1/10volume 2M sodium acetate and 2.5 volumes of 99 percent ethanol andincubating at -20° C. for 20 minutes. Centrifugation for 10 minutes at10,000xg yielded a pellet of completely double stranded DNA includingthe gene coding for HBxAg.

4. Plasmid DNA Analysis

The presence of HBV DNA in the clones was confirmed using the Southerntransfer and hybridization technique. Southern (1975), Mol. Biol.,98:503. 10 Mg of plasmid DNA from each clone isolated as above wasdigested with the BamHI (50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 1 mM dithiothreitol). The reaction mixture was electorophoresedin an 0.6 percent agarose horizontal slab gel at 100 amperes for 2 hours(0.04M Tris-acetate, 0.002M EDTA).

The DNA in the gel was denatured by soaking the gel in several volumesof 1.5M NaCl and 0.5M NaOH for 1 hour at room temperature with constantstirring or shaking. In some instances, the DNA in the gel washydrolyzed by acid depurination prior to alkali denaturation by soakingthe gel twice for 15 minutes in 0.25M HCl at room temperature. Afterdenaturation, the gel was neutralized by soaking in several volumes of asolution of 1M Tris-HCl (pH 8.0) and 1.5M NaCl for 1 hour at roomtemperature with constant shaking.

The DNA in the gel was transferred onto nitrocellulose essentially asdescribed in Maniatis et al. (1982), J. Molecular Cloning, Cold SpringHarbor. Briefly, nitrocellulose (Catalogue No. BA85, Schleicher &Schuel, Ohio) was placed on top of the gel and several layers of Whatman3MM paper are placed over the nitrocellulose. This sandwich was thenplaced gel side down on a Whatman 3MM wick whose ends were immersed in abuffer containing 0.9M NaCl and 0.09M sodium citrate. The capillarymovement of the buffer thereby transferred the DNA from the gel to thenitrocellulose. The DNA was fixed to the nitrocellulose by baking at 80°C. for 2 hours under vacuum.

The plasmid DNA on the nitrocellulose prepared above (Southern filters)was probed for the presence of HBV DNA fragments using the procedure ofManiatis et al., supra.

Briefly, the Southern filters were prehybridized by soaking inprehybridization fluid 0.9M NaCl, 0.09 M sodium citrate, 0.5 percent SDS(sodium dodecyl sulfate), 100 mg/ml denatured salmon sperm DNA and 5XDenhardt's Solution (containing, per liter thereof, 1 g Ficoll, 1 gpolyvinylpyrrolidone, and 1 g BSA Fraction V) for 4 hours at 68° C.

Hybridization was performed in a heat-sealable plastic bag with justenough hybridization solution to keep the Southern filter wet (50microliters/cm² of filter). Hybridization solution contained 0.9M NaCl,0.09M sodium citrate, 0.01M EDTA, 5X Denhardt's solution, 0.5 percentSDS, 100 micrograms/ml denatured salmon sperm DNA and 5×10⁷ cmp of theradioactively labeled HBV DNA prepared above. The Southern filter wasincubated in the hybridization solution for 12 hours at 68° C.

After washing the filter for 2 hours (0.3M NaCl, 0.03M sodium citrate),X-ray film (XRP-1, Kodak) was exposed to the filter to obtain anautoradiographic image.

Three recombinant plasmids were isolated using the above procedure anddesignated AM6, AM7 and AM1. AM6 contained pBR322 DNA and the entire HBVgenome. AM7 contained pBR222 DNA and a 1350 bp HBV DNA fragment. AM1contained pBR322 DNA and an 1850 bp HBV DNA fragment including the genecoding for HBxAg.

5. Construction of a Replication Plasmid Containing an SV40/HBxAgExpression Vector DNA

Defective SV40 virus obtained from Dean Hamer, supra, was subjected toBamHI and EcoRI cleavage in a buffer containing 50 mM NaCl, 10 mMTris-HCl (pH7.5), 10 mM MgCl₂, and 1 mM dithiothreitol. Plasmid pBR322DNA was subjected to the double digestion discussed before so as to formtermini complementary to the SV40 DNA so cleaved. The cleavage productsof each of these digestions were separated and purified by cesiumchooride density gradient centrifugation. Tanaka et al. (1975), J.Bact., 121:354-362. The 4492 bp SV40 fragment and 3987 bp pBR322fragment so isolated were subjected to DNA ligation with T4 DNA ligasein a buffer containing 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl₂, 10 mMdithiothreitol, and 0.4 mM ATP to form pBRSV (FIG. 4).

The ligation reaction product obtained in the above reaction is acircular DNA derived from the linearized pBR322 3987 bp and SV40 4492 bpfragments joining at their complementary EcoRI and BamHI termini. Thiscircular plasmid was then linearized by cleavage at the BamHIrestriction site formed during ligation creating BamHI cohesive terminiat each end.

An 1850 bp HBV DNA fragment including the gene coding from HBxAg wasisolated by subjecting plasmid AM6 isolated above to digestion withBamHI in a buffer containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mMMgC1₂, and 1 mM dithiothreitol. The reaction mixture was subjected toelectriphoesis at 100 amperes for 2 hours using 0.8 percent agarose andthe 1850 pb band was cut out and melted at 68° C. for 15 minutes. 1/10Volume of 3M sodium acetate was added, and the resulting solution wassubjected to the deproteinization procedure described before.

The HBxAg gene-containing HBV DNA fragment thus isolated had BamHIcomplementary termini. This DNA fragment was then ligated to the pBR-SVprepared above using T4 DNA ligase in a buffer containing 66 mM Tris-HCl(pH 7.6), mM MgCl₂, 10 mM dithiothreitol, and 0.4 mM ATP.

The product of the above ligation reaction was a circular plasmid DNAdesignated SVAM191. It contained, in clockwise order, a 4492 bp SV40 DNAfragment from its BamHI site at base position 2468 to its EcoRI site atbase position 1717, a 3987 bp pBR222 DNA fragment from its EcoRI site atbase position 0 to the BamHI site at base position 375 and an 1850 bpHBV DNA fragment from its BamHI site at base position at 1400 to theBamHI site at base position 28 plasmid SVAM191 is shown schematically inFIG. 4.

SVAM191 was introduced into E. coli HB101 using the transformationprocedure described above. Since these transformed E. coli were alsoampicillin resistant and tetracycline sensitive, they were subjected tothe same isolation procedure described before for the E. coli cloningvector. The procedures for production, isolation, purification andanalysis described before were employed to obtain milligram quantitiesof substantially pure SVAM191 DNA.

6. Construction of SV40/HBxAg Expression Vector

A vector capable of inducing the production of HBxAg in eucaryotic cellswas made by excising a portion of SVAM191 DNA. This was accomplished bydigesting SVAM191 DNA with HaeII endonucelose in a buffer containing 10mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and 1 mM dithiothreitol to cleaveSVAM191 at the HaeII sites at base position 767 in the SV40 DNA regionand base position 1437 in the HBV DNA region.

The two DNA fragments obtained from the above reaction were separated bythe electrophoretic agarose gel procedure described before. The largerfragment so isolated containing only SV40 and HBV DNA was circularizedby subjecting its HaeII complementary termini to the T4 DNA ligationprocedure described before.

The circular DNA expression vector formed above was designated SVHBV-3(FIGS. 4 and 5). It contains expression control elements from SV40 and agene coding for HBxAg from HBV.

7. Transfection of Eucaryotic Host Cells with SVHBV-3

The BSC-1 African Green Monkey kidney cell line (ATCC CCL 26; obtainedfrom Dr. G. B. Thornton, Johnson & Johnson Biotechnology Center, Inc.,La Jolla, Calif.), a permissive host for SV40, was chosen fortransfection with SVHBV-3. Confluent monolayers of about 10⁷ cells wereobtained by culturing at 37° C. in Eagle's minimal essential medium(EMEM) supplemented with 10 percent fetal calf serum, 100 units ofpenicillin/ml, 100 micrograms/ml streptomycin and 3 mM L-glutamine. Themonolayers were infected with vector SVHBV-3 DNA in the presence ofDEAE-dextran as described by Ganem et al. (1976), J. Mol. Biol.,101:57-83.

One flask was infected with 1.6 micrograms of the recombinant SVHBV-3together with 0.05 micrograms of SV40 tsA₂₃₉ DMA as helper. Controlsreceived equivalent amounts of the vector helper DNAs alone. Thecultures were incubated at 40° C. for 12 days, and were then lysed byfreeze-thawing, and stored at -70° C.

Cellular proteins were extracted from the freeze-thawed cel powdersobtained above by first adding 2 mg of cell powder to 1 ml of proteinextraction buffer [2 percent SDS, 10 percent glycerol, 0.08M Tris-HCl(pH 6.8), 2 mM phenyl methyl sulphonyl fluoride, 0.1M dithiothreitol,0.001 percent bromphenol blue]. The solution was then boiled for 5minutes, centrifuged at 15,000 rpm for 10 minutes and the supernatantscollected therefrom.

An amount of supernatant sufficient to provide 100 micrograms of sampleprotein was then subjected to SDS-polyacrylamide gel electrophoresis inthe Western Blot procedure described below.

8. Polypeptide Syntheses

The polypeptides of this invention were chemically synthesized bysolid-phase methods as described in Merrifield et al. (1963), J. Am.Chem. Soc., 85:2149; Merrifield et al. (1970), A. Rev. Biochem.,39:841-866 and Houghten et al. (1980), Int. J. Peptide Prot. Res.,16:311-320. The relatively short polypeptides used herein correspond toantigenic determinants of HBxAg.

FIG. 6 shows the 154 amino acid residue sequence of HBxAg. The aminoacid residue sequences of the preferred synthetic polypeptides describedherein (99, 100 and 142) are also shown in FIG. 6. The composition ofboth synthetic polypeptides was confirmed by amino acid analysis.

Generally, an immunogen or synthetic polypeptide is made by the steps ofproviding a plurality of suitably protected amino acids that correspondto the amino acid residues of an antigenic determinant domain of HBxAg,and synthesizing those amino acids into a polypeptide that has an aminoacid residue sequence corresponding to the polypeptide amino acidresidue sequence of that antigenic determinant. The produced syntheticpolypeptide can be used to produce an inoculum, usually by linking it toa carrier to form a conjugate and then dispersing an effective amount ofthe conjugate in a physiologically tolerable diluent.

The oolypeptides are preferably synthesized according to theabove-referenced solid phase methods using a cysteine resin. SeeMerrifield et al., J. Am. Chem. Soc., supra. Using that method, thealpha-amino group of each added amino acid is typically protected by atertiary-butoxycarbonyl (t-BOC) group prior to the amino acid beingadded into the growing polypeptide chain. The t-BOC group is thenremoved prior to addition of the next amino acid to the growingpolypeptide chain. The side chains on individual amino acids areprotected as follows: Arg-tosyl; Ser-, Thr-, Glu- and Asp-O-benzyl;Tyr-O-bromobenzyloxy carbamyl; Trp-N-formyl; S-methoxybenzyl forcysteine; 2-chlorobenzoxycarbonyl for lysine; and dinitrophenyl forhistidine. When asperigine is used, an equal molar amount ofN-hydroxy-benztriazole is added with the protected amino acid anddimethyl formamide (DMF) is used as the coupling solvent. The N-formylgroup on the Trp residues is removed after cleavage of the polypeptidefrom the resin support by treatment with 1.0 molar ammonium bicarbonateat a polypeptide concentration of 1.0 milligram/milliliter for 16 hoursat the room temperature. Yamashiro et al. (1973), J. Org. Chem.,38:2594-2597. The efficiency of coupling at each step can be monitoredwith ninhydrin or picric acid, and is preferably greater than 99 percentin all cases. See Gisin (1972), Anal. Chem. Acta, 58:248-249; and Kaiser(1980), Anal. Biochem., 34:595-598.

After preparation of a desired polypeptide, a portion of the resulting,protected polypeptide (about 1 gram) is treated with two milliliters ofanisole, and anhydrous hydrogen flouride, about 20 milliliters, iscondensed into the reaction vessel at dry ice temperature. The resultingmixture is stirred at about 4° C. for about one hour to cleave theprotecting groups and to remove the polypeptide from the resin. Afterevaporating the hydrogen flouride at a temperature of 4° C. with astream of N₂, the residue is extracted with anhydrous diethyl etherthree times to remove the anisole, and the residue is dried in vacuo.

The vacuum dried material is extracted with 5% aqueous acetic acid (3times 50 milliliters) to separate the free polypeptide from the resin.The extract-containing solution is lyophilized to provide a monomericunoxidized polypeptide.

Briefly, as a generalized procedure for each polypeptide, 4 milligramsof KLH in 0.25 millileters of 10 millimolar sodium phosphate buffer (pH7.2) is reacted with 0.7 milligrams of MBS dissolved in DMF, and theresulting admixture is stirred for 30 minutes at room temperature. TheMBS solution is added dropwise to ensure that the local concentration ofDMF was not too high, as KLH is insoluble at DMF concentrations of about30% or higher. The reaction product, KLH-MB, is passed through achromatography column prepared with Sephadex G-25 (Pharmacia FineChemicals, Piscataway, NJ) equilibrated with 50 millimolar sodiumphosphate buffer (pH 6.0) to remove free MBS. KLH recovery from peakfractions of the column eluate, monitored at 280 nanometers, istypically approximately 80%.

The KLH-MB so prepared is then reacted with 5 milligrams of polypeptidedissolved in 1 milliliter of buffer. The pH value of the resultingreaction composition is adjusted to 7-7.5, and the reaction compositionis stirred at room temperature for 3 hours to provide apolypeptide-carrier conjugate.

9. Western Blotting

The anti-polypeptide antibodies (anti-99, anti-100 and anti-142) wereexamined using the Western Blot technique to confirm their predictedspecificity for HBxAg, and to confirm the expression of the substantialpolypeptide portion of HBxAg in transfected cells. The cellular proteinsincluding HBxAg were separated by 12.5 percent SDS-polyacrylamide gelelectrophoresis. See Laemmli (1970), Nature, 277:680-685; and Towbin etal. (1979), Proc. Natl. Acad. Sci., USA, 76:4350-4354.

Proteins were electrophoretically transferred to nitrocellulose(Schleicher & Schuel, Catalogue No. BA85) as described by Towbin et al.,supra, using an electroblot apparatus (E.C. Apparatus Corp. ofJacksonville, Fla.) with a buffer consisting of 25 mM Tris-Base, 192 mMglycine, 20 percent methanol and 0.1 percent SDS (pH 8.3). Following thetransfer, the nitrocellulose was blocked in BLOTTO [Bovine LactoTransfer Technique Optimizer, Johnson et al. (1983), J. Exp. Med.,159:1751-1756; 5% (w/v) non-fat dry milk; 0.01% anti-foam A (Sigma,Catalogue No. A5758), and 0.0001% merthiolate (Sigma, Catalogue No.5125) in PBS at pH 7.2] to reduce non-specific binding. The blots werereacted with 100 microliters of antipeptide antibody in 10 ml of BLOTTOfor 3 hours and then washed 3 times for 1 hour with 50 ml of freshBLOTTO.

Anti-polypeptide antibodies bound to vector-specific protein weredetected by reacting the blots with 20 microliters of ¹²⁵ I-labeledStaphylococcus aureus protein A in 10 milliliters of BLOTTO for 1 hour.The blots were then washed in 50 milliliters of fresh BLOTTO for 15minutes 4 times and then under a continuous flow of water for 20minutes.

10. ¹²⁵ I Labeling of Hepatoma Cell Extracts

Monolayers of the human hepatoma-derived cell line PLC/PRF/5 known tocontain integrated sequences of HBV [Alexander et al. (1976), AfricanMed. J., 50:21-24; ATCC CRL 8024] were lyopholized. The cell powder wasdissolved in phosphate-buffered saline (PBS) to achieve a 1 mg/mlprotein concentration. After centrifugation at 10,000xg to removecellular debris, 50 microliters of the above solution were admixed with75 microliters of RIPA [0.15M NaCl, 10 mM sodium phosphate (pH 7.5), 1%Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS] and 20 microliters of0.2M sodium phosphate (pH 7.5). In one study, the thus solubilized cellprotein was labeled with 5 milliCuries of ¹²⁵ I using the chloramine Treaction. In the study shown in FIG. 7, the cell lysates were labeledwith 3 microCuries of ¹²⁵ I using the same reaction.

The above reaction mixture was run through the Sephadex G-25 (PharmaciaFine Chemicals, Piscataway, NJ) column washed with PBS. The radiolabeledpeak fraction (9×10⁶ cpm/ml) obtained was pre-incubated with normalrabbit serum to remove nonspecific binding. Briefly, 20 microliters of apeak fraction were admixed with 1.0 ml of RIPA, 20 microliters Trasylol(a trademark for a protinin Sigma, February, 1984 Catalogue page 163)and 100 microliters of NRS. After incubation for 1 hour at zero° C. 500microliters of formalin-fixed Staphylococcus aureus (Staph A;Calbiochem, La Jolla, CA) cells were admixed, and the admixture wasincubated at zero °C. for 30 minutes. The Staph A precipitate wasremoved by centrifugation at 10,000xg for 10 minutes, and thesupernatant was recovered.

11. Immunoprecipitations (IP)

Rabbit anti-polypeptide antiserum against polypeptide 99 (anti-99) wasreacted with the above obtained radioiodine-labeled cell proteins.Briefly, 10 microliters of anti-99 was admixed with 2×10⁶ cpm of labeledextract and incubated for 1 hour at zero °C. 40 Microliters of Staph Awere then admixed and incubated for 30 minutes at zero °C. The Staph Aprecipitate was removed by centrifugation at 10,000xg for 10 minutes,and the pellet was recovered. The pellet was resuspended in 1 ml of RIPAand centrifuged as above. The resulting pellet was resuspended in 1.0 mlLiCl solution (100 mM Tris HCl, 500 mM LiCl), and was again centrifuged.The LiCl solution treatment was repeated and the pellet recovered.

The above-obtained pellet was resuspended in 50 microliters of samplebuffer and boiled for 3 minutes. The mixture was centrifuged at 10,000xgfor 1 minute and the supernatant recovered, and was subjected to 12.5percent SDS-poly-acrylamide gel electrophoresis. The above gels wereexposed to XRP-1 X-ray film to obtain an autoradiograph.

12. Preparation and Assay of Chimpanzee and Human Liver Cell Extracts

Liver samples from two HBV chronically infected chimpanzees and a human,and from normal chimpanzee and human (HBV sero-negative) livers werequick frozen in liquid nitrogen, and were ground to a powder. Thepowders were admixed into sample buffer [0.0625M Tris-HCl (pH 6.8), 2%SDS, 10% gylcerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue] andboiled for 5 minutes. The mixture was centrifiged at 10,000xg for 30minutes to remove cell debris. The samples were then subjected toWestern Blot analysis as described before using 75 micrograms of proteinper gel lane.

13. Identification of Anti-HBxAg Antibodies in Human Sera

20 Micrograms per lane of SVHBV-3 transfected BSC-1 cell extracts weresubjected to SDS-PAGE on 12 percent acrylamide gels, and the separatedproteins were transferred electrophoretically to nitrocellulose sheetsas before-described. The resulting nitrocellulose sheets were admixedwith 1:50 dilutions of serum from six humans that had beeen diagnosed ashaving HBV-related infections, including a symptomatic carrier and apatient with a hepatacellular carcinoma. Control sheets were admixedwith rabbit-anti-polypeptide antibodies of this invention.

The nitrocellulose-protein serum admixtures were maintained for 2 hoursat room temperature. The sheets were then rinsed and admixed with a1:200 dilution of goat anti-human or anti-rabbit antibodies linked tohorseradish peroxidase, as was appropriate. That admixture wasmaintained for a time period of 1 hour for bound anti-X antibodies toreact with the appropriate anti-antibodies. The sheets were washed andthen developed with 4-chloro-1-naphthol, as previously described. Theserum from the patient with heptacellular carcinoma exhibited strongimmunoreactivity with the approximately 24,000 dalton polypeptideexpressed by the SVHBV-3 transfected cells.

14. Cell Lines and Tissue Samples

PLC/PRF/5 and HepG2 cells were provided by Dr. D. Milich, Department ofBasic & Clinical Research, Scripps Clinic and Research Foundation(Scripps), La Jolla, CA. Chimpanzee liver tissue samples were providedby Drs. R. Purcell and P. Kaplan of the National Institute of Allergyand Infectious Diseases, Bethesda, MD, and Ortho Diagnostics, Inc.,Raritan, NJ, respectively. Human liver tissue samples were provided byDrs. F. Chisari, J. Dienstag and A. Yu of Scripps, Department ofMedicine, Harvard University, Boston, MA, and Department of Pediatrics,University of California-San Diego, La Jolla, CA, respectively.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the novel conceptsof the invention.

What is claimed is:
 1. An antigenic synthetic polypepide selected fromthe group consisting of polypeptides represented by the formulae,written from left to right and in the direction of amino-terminus tocarboxy-terminus:(i)Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp; (ii)Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val; and(iii) Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala.
 2. The polypeptideaccording to claim 1 further including a cysteine residue at the amino-or carboxy-terminus of said polypeptide.
 3. A water-soluble orwater-dispersible antigenic polymer containing a plurality of joinedsynthetic polypetide repeating units bonded together by oxidizedcysteine residues, said repeating units being represented by a formula,written from left to right and in the direction from amino-terminus tocarboxy-terminus, selected from the group consistingof:Cys-Leu-Ser-Ala-Met-Ser-Thr-Thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp-Cys;Cys-Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Glu-Glu-Ile-Arg-Leu-Lys-Val-Cys;and R¹ -Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala-R² wherein each ofR¹ and R² is a cysteine residue, with the proviso that only one of R¹and R² is present.
 4. A receptor molecule that includes an antibodycombining site capable of immunoreacting with an antigenic syntheticpolypeptide selected from the group consisting of polypeptidesrepresented by the formulae written from left to right and in thedirection of amino-terminus to carboxy-terminus:(i)Leu-Ser-Ala-Met-Ser-Thr-thr-Asp-Leu-Glu-Ala-Tyr-Phe-Lys-Asp; (ii)Leu-Phe-Lys-Asp-Trp-Glu-Glu-Leu-Gly-Gly-Glu-Ile-Arg-Leu-Lys-Val; and(iii) Ala-Pro-Ala-Pro-Cys-Asn-Phe-Phe-Thr-Ser-Ala.